Detection of human umbilical cord tissue derived cells

ABSTRACT

The invention relates to methods for detecting allogeneic therapeutic cells (such as human umbilical cord tissue-derived cells (hUTC)) in blood. The methods includes the steps of identifying one or more one or more markers positive for allogeneic therapeutic cells (e.g. hUTC) and one or more markers positive for human peripheral blood mononuclear cells (PBMC); providing a blood sample from a patient that has been treated with allogeneic therapeutic cells (e.g. hUTC), analyzing the sample using an assay method to detect one or more markers positive for PBMC and one or more markers positive for allogeneic therapeutic cells (e.g. hUTC); and distinguishing between the PBMC and one or more markers positive for allogeneic therapeutic cells (e.g. hUTC). In one embodiment, the cells are hUTC and the markers positive of hUTC include CD10 and/or CD13 and the one or more markers positive for PBMC includes CD45.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application61/579,710 (filed on Dec. 23, 2011) which is incorporated by referencein its entirety.

FIELD OF THE INVENTION

The invention relates to methods of detecting allogeneic therapeuticcells, such as e.g. human umbilical cord tissue-derived cells, in asample from a patient that treated with the therapeutic cells.

BACKGROUND OF THE INVENTION

Allogeneic cell therapies are a promising new technology for thetreatment of a number of unmet medical needs. However, cell therapiesare unique products and pose some unique challenges in the developmentprocess. One specific example of this technology is the development ofhuman umbilical cord tissue-derived cells (“hUTC”) for a number ofclinical indications. Following administration of hUTC to subjects,measuring the presence and/or the number of cells detected in thesubject's blood is desirable information relevant to thepharmacokinetics of hUTC as a cell therapy product. However, this posesa challenge since hUTC may have characteristics that are similar tocells gathered from the blood of the subject. Therefore, it is necessaryto distinguish hUTC from other cells.

Clinical studies during drug development include pharmacokinetic studiesto examine parameters of absorption, distribution, metabolism, andexcretion of the drug in vivo. An important element of thepharmacokinetic studies is to determine the level of exposure of a drugin subjects. Typically, this is done through analysis of drug levelsfrom blood samples; exposure levels are evaluated relative to efficacyand safety outcomes. In the case of a cell therapy product, such ashUTC, studying the bio-distribution or pharmacokinetics of hUTC inclinical trials presents a challenge because there is no establishedmethod to distinguish hUTC (or other cell products) from the subjects'own cells. Therefore, it is difficult to determine the bioavailabilityof hUTC.

Presently accepted approach for determining if allogeneic therapeuticcells (e.g. hUTC) are present in the circulation requires the use ofallogeneic therapeutic cells (e.g. hUTC) from a male donor andintravenous transfusion into female subjects. See e.g. Bader., P. etal., “How and when should we monitor chimerism after allogeneic stemcell transplantation?” Bone Marrow Transplantation, 2005; 35: 107-119;see also Durnam, D M et al., “Analysis of the origin of marrow cells inbone marrow transplant recipients using a Y-chromosome-specific in situhybridization assay,” Blood, 1989; 74: 2220-2226. Real time PCR is usedto detect the Y chromosome in a sample of the subject's blood and theresults provide a relative quantification or binary signal to indicatethe presence or absence of allogeneic therapeutic cells (e.g. hUTC) inthe blood sample. See Bader, P. et al. This approach necessitatesexcluding female cell (e.g. hUTC) donors and male subjects from analyseson pharmacokinetics of hUTC in clinical studies. Therefore, thereremains a need for a method for detecting allogeneic cells, such as e.g.UTC, in patients after administration of the cells.

SUMMARY OF THE INVENTION

The invention provides for detecting the presence of allogeneictherapeutic cells in a sample of human peripheral blood mononuclearcells. The invention allows for the detection of such therapeutic cellswithout being constrained by the therapeutic cells karyotype (XY vs. XX)and recipient (patient) gender.

One embodiment of the invention is a method of detecting allogeneictherapeutic cells in blood comprising: (a) assaying allogeneictherapeutic cells and human peripheral blood mononuclear cells toidentify one or more markers positive for allogeneic therapeutic cellsand one or more markers positive for patient peripheral bloodmononuclear cells; (b) providing a blood sample from a patient that hasbeen treated with allogeneic therapeutic cells; (c) analyzing the sampleusing an assay method to detect one or more markers positive for patientperipheral blood mononuclear cells and one or more markers positive forallogeneic therapeutic cells; and (d) distinguishing between the patientperipheral blood mononuclear cells and allogeneic therapeutic cellsbased on the detection of one or more markers positive for patientperipheral blood mononuclear cells and one or more markers positive forallogeneic therapeutic cells. In one embodiment, the one or morepositive markers for patient peripheral blood mononuclear cellscomprises CD45. Alternatively, the method for detecting allogeneictherapeutic cells in blood includes: (a) assaying allogeneic therapeuticcells and human peripheral blood mononuclear cells to identify one ormore markers positive for allogeneic therapeutic cells and one or moremarkers positive for patient peripheral blood mononuclear cells; (b)comparing the one or more markers positive for allogeneic therapeuticcells and the one or more markers positive for patient peripheral bloodmononuclear cells to identify one or more unique markers whichdistinguishes the allogeneic therapeutic cells from the patientperipheral blood mononuclear cells; (c) providing a blood sample from apatient that has been treated with allogeneic therapeutic cells; (d)analyzing the sample using an assay method to detect the one or moreunique markers positive for the allogeneic therapeutic cells; and (e)distinguishing between the patient peripheral blood mononuclear cellsand allogeneic therapeutic cells based on the detection of the one ormore unique markers.

The methods may be suitable to detect any allogeneic therapeutic cellsof interest. For example, the allogeneic therapeutic cells may beselected from the group consisting of human umbilical cordtissue-derived cells, human umbilical cord blood-derived cells,placental-derived cells, mesenchymal stem cell derived cells, livercells, pancreatic islet cells, cardiomyocytes, and insoluble collagenousbone matrix cells. The methods may also be used to detect two or moredifferent types of allogeneic therapeutic cells in a blood sample.

A variety of different assay methods may be used for these methods, suchas e.g. flow cytometry, ELISA, immunohistochemistry, nucleic aciddetection, PCR, and combinations thereof. Additionally, the methods mayinclude an enrichment step between steps (a) and (b). The enrichmentstep may include magnetic capture technology. The step of distinguishingincludes differentiating between allogeneic therapeutic cellsadministered to the patient and peripheral blood mononuclear cells fromthe patient. The patient may be a human, non-human primate, mouse, rat,hamster, guinea pig, dog, or pig.

The invention also provides for kits, which may be used use in themethod of detecting allogeneic therapeutic cells in a blood sample. Thekits may include a marker profile having one or more markers positiveallogeneic therapeutic cells and one or more markers positive forpatient peripheral blood mononuclear cells.

Another embodiment of the invention is a method of detecting humanumbilical cord tissue-derived cells in blood including: (a) assayinghuman umbilical cord tissue-derived cells and human peripheral bloodmononuclear cells to identify one or more markers positive for humanumbilical cord tissue-derived cells and one or more markers positive forhuman peripheral blood mononuclear cells; (b) providing a blood samplefrom a patient that has been treated with human umbilical cordtissue-derived cells; (c) analyzing the sample using an assay method todetect one or more markers positive for patient peripheral bloodmononuclear cells and one or more markers positive for human umbilicalcord tissue-derived cells; and (d) distinguishing between the patientperipheral blood mononuclear cells and human umbilical cordtissue-derived cells based on the detection of one or more markerspositive for patient peripheral blood mononuclear cells and one or moremarkers positive for human umbilical cord tissue-derived cells.Alternatively, the method of detecting human umbilical cordtissue-derived cells in blood includes: (a) assaying human umbilicalcord tissue-derived cells and human peripheral blood mononuclear cellsto identify one or more markers positive for human umbilical cordtissue-derived cells and one or more markers positive for patientperipheral blood mononuclear cells; (b) comparing the one or moremarkers positive for human umbilical cord tissue-derived cells and theone or more markers positive for human peripheral blood mononuclearcells to identify one or more unique markers which distinguishes theumbilical cord tissue-derived cells from the patient peripheral bloodmononuclear cells; (c) providing a blood sample from a patient that hasbeen treated with human umbilical cord tissue-derived cells; (d)analyzing the sample using an assay method to detect one or more uniquemarkers positive for human umbilical cord tissue-derived cells; and (e)distinguishing between the patient peripheral blood mononuclear cellsand human umbilical cord tissue-derived cells based on the detection ofthe one or more unique markers. The patient may be a human, non-humanprimate, mouse, rat, hamster, guinea pig, dog, or pig.

In one embodiment, the positive marker for patient peripheral bloodmononuclear cells is CD45 and the positive marker for human umbilicalcord tissue-derived cells is CD10. In another embodiment, markers forpatient peripheral blood mononuclear cells and for human umbilical cordtissue-derived cells include one or more of CD10, CD13, NRP1, CD45,LAMP1, DKK3, NRP1, or LAMB1. The method may employ a variety of assaytechniques including flow cytometry, ELISA, immunohistochemistry,nucleic acid detection, PCR, and combinations thereof. The one or moreunique markers positive for human umbilical cord tissue-derived cellsmay be one or more of CD10, CD13, NRP1, LAMP1, DKK3, NRP1, LAMB1 andcombinations thereof.

The method may further comprise performing an enrichment step betweensteps (a) and (b). The enrichment step may be magnetic capturetechnology. Another embodiment of the invention is a kit for use in themethod of detecting human umbilical cord tissue-derived cells in bloodcomprising a marker profile having one or more markers positive forhuman umbilical cord tissue-derived cells and one or more markerspositive for human peripheral blood mononuclear cells. The inventionalso provides for systems for use with the methods of the invention.

Another embodiment of the invention is a method of detecting humanumbilical cord tissue-derived cells in blood including the steps of:assaying human umbilical cord tissue-derived cells and human peripheralblood mononuclear cells to identify one or more markers positive forhuman umbilical cord tissue-derived cells and one or more markerspositive for human peripheral blood mononuclear cells; providing a bloodsample containing human umbilical cord tissue-derived cells; isolatingthe human umbilical cord tissue-derived cell/peripheral bloodmononuclear cell fraction from the blood sample; analyzing the humanumbilical cord tissue-derived cell/peripheral blood mononuclear cellfraction by flow cytometry for CD45 as a positive marker for peripheralblood mononuclear cell and CD10 or CD13 as a positive marker for humanumbilical cord tissue-derived cells and detecting the presence of thehuman peripheral blood mononuclear cells and human umbilical cordtissue-derived cells based on the detection of CD45 as a marker positivefor human peripheral blood mononuclear cells and CD10 or CD13 as amarker positive for human umbilical cord tissue-derived cells.

The step of analyzing may include analysis of the human umbilical cordtissue-derived cell/peripheral blood mononuclear cell fraction by flowcytometry for CD45 as a positive marker for peripheral blood mononuclearcell and CD13 as a positive marker for human umbilical cordtissue-derived cells. Alternatively the step of analysis may includeanalysis of the human umbilical cord tissue-derived cell/peripheralblood mononuclear cell fraction by flow cytometry for CD45 as a positivemarker for peripheral blood mononuclear cell and CD10 as a positivemarker for human umbilical cord tissue-derived cells. The method mayalso further include performing an enrichment step prior to analyzingthe human umbilical cord tissue-derived cell/peripheral bloodmononuclear cell fraction such as e.g. magnetic capture technology.

Another embodiment of the invention is a method of detecting humanumbilical cord tissue-derived cells in blood including the steps of:providing a blood sample containing human umbilical cord tissue-derivedcells; isolating the human umbilical cord tissue-derived cell/peripheralblood mononuclear cell fraction from the blood sample; removing anyplasma; analyzing the human umbilical cord tissue-derivedcell/peripheral blood mononuclear cell fraction by flow cytometry forCD45 as a positive marker for peripheral blood mononuclear cell and CD13as a positive marker for human umbilical cord tissue-derived cells anddetecting the presence of the human peripheral blood mononuclear cellsand human umbilical cord tissue-derived cells based on the detection ofCD45 as a marker positive for human peripheral blood mononuclear cellsand CD13 as a marker positive for human umbilical cord tissue-derivedcells. The method may also further include performing an enrichment stepprior to analyzing the human umbilical cord tissue-derivedcell/peripheral blood mononuclear cell fraction such as e.g. magneticcapture technology. The method may also further include detecting forCD10 as a marker positive for human umbilical cord tissue-derived cells.

Human umbilical cord tissue-derived cells are isolated from humanumbilical cord tissue substantially free of blood, are capable ofself-renewal and expansion in culture, and have the potential todifferentiate. In one embodiment, the human umbilical cordtissue-derived cells are isolated from human umbilical cord tissuesubstantially free of blood, are capable of self-renewal and expansionin culture, have the potential to differentiate, and have the followingcharacteristics: (1) express CD10, CD13, CD44, CD90, and HLA-ABC; (2) donot express CD31, CD34, CD45, HLA-DR and CD117, and (3) do not expresshTERT or telomerase. Alternatively, the human umbilical cordtissue-derived cells may be isolated from human umbilical cord tissuesubstantially free of blood, are capable of self-renewal and expansionin culture, have the potential to differentiate, and have the followingcharacteristics: (1) express CD10, CD13, CD44, CD90, and HLA-ABC; (2) donot express CD31, CD34, CD45, HLA-DR and CD117; (3) do not express hTERTor telomerase; (4) express oxidized low density lipoprotein receptor 1,reticulon, chemokine receptor ligand 3, and/or granulocyte chemotacticprotein; and (4) express, relative to a human fibroblast, mesenchymalstem cell, or iliac crest bone marrow cell, increased levels ofinterleukin 8 or reticulon 1.

Another embodiment of the invention is method of detecting humanumbilical cord tissue-derived cells in blood including the steps of: (a)providing a blood sample from a patient that has been treated with humanumbilical cord tissue-derived cells; (b) analyzing the sample using anassay method to detect one or more markers positive for human peripheralblood mononuclear cells and one or more markers positive for humanumbilical cord tissue-derived cells; and (c) distinguishing between thehuman peripheral blood mononuclear cells and one or more markerspositive for human umbilical cord tissue-derived cells. In oneembodiment, the positive marker for human peripheral blood mononuclearcells is CD45 and the positive marker for human umbilical cordtissue-derived cells is CD10. The step of analyzing may utilize flowcytometry, ELISA, immunohistochemistry, nucleic acid detection, and/orPCR. The method may further include the performing an enrichment stepbetween steps (a) and (b). The enrichment step may be magnetic capturetechnology.

Yet another embodiment of the invention is a method of detecting humanumbilical cord tissue-derived cells in blood including the steps of: (a)providing a blood sample containing human umbilical cord tissue-derivedcells; (b) isolating the human umbilical cord tissue-derivedcell/peripheral blood mononuclear cell fraction from the blood sample;and (c) analyzing the human umbilical cord tissue-derivedcell/peripheral blood mononuclear cell fraction by flow cytometry forCD45 as a positive marker for peripheral blood mononuclear cell and CD10as a positive marker for human umbilical cord tissue-derived cells.

An alternate embodiment of the invention is a method of detecting humanumbilical cord tissue-derived cells in blood comprising the steps of:providing a blood sample containing human umbilical cord tissue-derivedcells; isolating the human umbilical cord tissue-derived cell/peripheralblood mononuclear cell fraction from the blood sample; removing anyplasma; and analyzing the human umbilical cord tissue-derivedcell/peripheral blood mononuclear cell fraction by flow cytometry forCD45 as a positive marker for peripheral blood mononuclear cell and CD13as a positive marker for human umbilical cord tissue-derived cells.

Other features and advantages of the invention will be apparent from thedetailed description and examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended figures. For the purpose of illustrating the invention, thefigures demonstrate embodiments of the present invention. It should beunderstood, however, that the invention is not limited to the precisearrangements, examples, and instrumentalities shown.

FIG. 1 lists the 24 samples and the age of the cell culture for eachsample used for Study 1 in Example 1. In particular, the Table at FIG.1A lists the 24 samples used for microarray analysis and the chart atFIG. 1B shows the age of the cell culture for each of these 24 samples.

FIG. 2A shows a comparative expression of PTPRC(CD45) gene in humanumbilical cord tissue-derived cells (hUTC) and in various types of bloodcells that comprise of human PBMC.

FIG. 2B shows a comparison of expression of MME (CD10) gene in hUTC andin various types of blood cells that comprise of human peripheral bloodmononuclear cell (PBMC).

FIG. 2C shows a comparison of expression of ANPEP (CD13) gene in hUTCand in various types of blood cells that comprises of human PBMC.

FIG. 3 shows RT-PCR analysis of select genes whose expression in hUTC ishigher than that of human PBMC.

FIG. 4A shows the results of a flow cytometry assay for the detection ofcell surface markers in hUTC. With reference to FIG. 4A, panes A1, B1,C1, D1, and E1 show the unstained controls. Pane A2 shows CD13 and 7AAD.Pane B2 shows CD10 and 7AAD. Pane C2 shows NRP1 and 7AAD. Pane D2 showsCD45 and 7AAD. Pane E2 shows LAMP1 and 7AAD.

FIG. 4B shows the results of a flow cytometry assay for the detection ofcell surface markers in human PBMC. With reference to FIG. 4B, panes A1,B1, C1, D1, and E1 show the unstained controls. Pane A2 shows CD13 and7AAD. Pane B2 shows CD10 and 7AAD. Pane C2 shows NRP1 and 7AAD. Pane D2shows CD45 and 7AAD. Pane E2 shows LAMP1 and 7AAD.

FIG. 4C shows the results for a flow cytometry assay for the detectionof the internal markers DKK3 and LAMP1 in PBMC and hUTC.

FIG. 5A shows the detection of hUTC in a mixture comprising of hUTC(ranging from 1,500-1,700 cells/ml) and human PBMC (1 million cells/ml)in the presence of 1 ml of human serum using flow cytometry.

FIG. 5B shows the detection of hUTC in a mixture comprising of hUTC(ranging from 1, 700 to 110,000 cells/ml) and human PBMC (1 millioncells/ml) in the presence of 1 ml of human serum using flow cytometry.

DETAILED DESCRIPTION

This application is directed to methods of detecting allogeneictherapeutic cells such as progenitor cells circulating in the blood of apatient (e.g. human). Progenitor cells or other engineered cells, whichare components of a cell therapy product, are being developed for anumber of clinical indications. These cells, such as e.g. hUTC, areadministered to the patient via e.g. intravenous administration. Thedisposition of these cells in circulating blood needs to be determinedin order to assess the pharmacokinetics of the cells as a cell therapyproduct and also to more accurately determine success of the therapy.However, human blood is composed of multiple types of blood cells, oneor more type of which express some proteins that may also be expressedin the progenitor or engineered cells. Moreover, it is important todevelop a sufficiently sensitive assay that can detect a small number ofthese cells (such as e.g. hUTC) in a large excess of cells in therecipient's blood. Detecting these cells in the presence of human blood,therefore, becomes a challenge that requires obtaining sufficientsensitivity and specificity. Thus, there is a need for an assay that canidentify and distinguish progenitor or engineered cells from human bloodcells.

I. Definitions

A “sample” as used herein, refers to any substance, which may containthe analyte of interest. A sample can be biological fluid, such as wholeblood or whole blood components including red blood cells, white bloodcells, platelets, serum and plasma, ascetic fluid, urine, cerebrospinalfluid, and other constituents of the body.

The cells which may be identified in a blood sample containingperipheral mononuclear cells using the methods of the invention aregenerally referred to as postpartum cells or postpartum-derived cells(PPDCs). The cells are more specifically “umbilicus-derived cells” or“umbilical cord-derived cells” (UDC), or “umbilical cord tissue-derivedcells” (UTC) or “human umbilical cord tissue-derived cells” (hUTC). Inaddition, the cells may be described as being stem or progenitor cells,the latter term being used in the broad sense. The term “derived” isused to indicate that the cells have been obtained from their biologicalsource and grown or otherwise manipulated in vitro (e.g., cultured in agrowth medium to expand the population and/or to produce a cell line).The in vitro manipulations of umbilical stem cells and the uniquefeatures of the umbilicus-derived cells of the present invention aredescribed in detail below.

Stem cells are undifferentiated cells defined by the ability of a singlecell both to self-renew and to differentiate to produce progeny cells,including self-renewing progenitors, non-renewing progenitors, andterminally differentiated cells. Stem cells are also characterized bytheir ability to differentiate in vitro into functional cells of variouscell lineages from multiple germ layers (endoderm, mesoderm andectoderm), as well as to give rise to tissues of multiple germ layersfollowing transplantation, and to contribute substantially to most, ifnot all, tissues following injection into blastocysts.

Stem cells are classified according to their developmental potential as:(1) totipotent; (2) pluripotent; (3) multipotent; (4) oligopotent; and(5) unipotent. Totipotent cells are able to give rise to all embryonicand extraembryonic cell types. Pluripotent cells are able to give riseto all embryonic cell types. Multipotent cells include those able togive rise to a subset of cell lineages, but all within a particulartissue, organ, or physiological system. For example, hematopoietic stemcells (HSC) can produce progeny that include HSC (self-renewal), bloodcell-restricted oligopotent progenitors, and all cell types and elements(e.g., platelets) that are normal components of the blood. Cells thatare oligopotent can give rise to a more restricted subset of celllineages than multipotent stem cells. Cells that are unipotent are ableto give rise to a single cell lineage (e.g., spermatogenic stem cells).

Stem cells are also categorized based on the source from which they areobtained. An adult stem cell is generally a multipotent undifferentiatedcell found in tissue comprising multiple differentiated cell types. Theadult stem cell can renew itself. Under normal circumstances, it canalso differentiate to yield the specialized cell types of the tissuefrom which it originated, and possibly other tissue types. An embryonicstem cell is a pluripotent cell from the inner cell mass of ablastocyst-stage embryo. A fetal stem cell is one that originates fromfetal tissues or membranes. A postpartum stem cell is a multipotent orpluripotent cell that originates substantially from extraembryonictissue available after birth, namely, the umbilical cord. These cellshave been found to possess features characteristic of pluripotent stemcells, including rapid proliferation and the potential fordifferentiation into many cell lineages. Postpartum stem cells may beblood-derived (e.g., as are those obtained from umbilical cord blood) ornon-blood-derived (e.g., as obtained from the non-blood tissues of theumbilical cord).

Various terms are used to describe cells in culture. “Cell culture”refers generally to cells taken from a living organism and grown undercontrolled conditions (“in culture” or “cultured”). A “primary cellculture” is a culture of cells, tissues, or organs taken directly froman organism(s) before the first subculture. Cells are “expanded” inculture when they are placed in a growth medium under conditions thatfacilitate cell growth and/or division, resulting in a larger populationof the cells. When cells are expanded in culture, the rate of cellproliferation is sometimes measured by the amount of time needed for thecells to double in number. This is referred to as “doubling time.”

The term “cell line” generally refers to a population of cells formed byone or more subcultivations of a primary cell culture. Each round ofsubculturing is referred to as a passage. When cells are subcultured,they are referred to as having been “passaged.” A specific population ofcells, or a cell line, is sometimes referred to or characterized by thenumber of times it has been passaged. For example, a cultured cellpopulation that has been passaged ten times may be referred to as a P10culture. The primary culture, i.e., the first culture following theisolation of cells from tissue, is designated P0. Following the firstsubculture, the cells are described as a secondary culture (P1 orpassage 1). After the second subculture, the cells become a tertiaryculture (P2 or passage 2), and so on. It will be understood by those ofskill in the art that there may be many population doublings during theperiod of passaging; therefore, the number of population doublings of aculture is greater than the passage number. The expansion of cells(i.e., the number of population doublings) during the period betweenpassaging depends on many factors, including, but not limited to, theseeding density, substrate, medium, growth conditions, and time betweenpassaging.

“Differentiation” is the process by which an unspecialized(“uncommitted”) or less specialized cell acquires the features of aspecialized cell, such as e.g. a nerve cell or a muscle cell. A“differentiated” cell is one that has taken on a more specialized(“committed”) position within the lineage of a cell. The term“committed”, when applied to the process of differentiation, refers to acell that has proceeded in the differentiation pathway to a point where,under normal circumstances, it will continue to differentiate into aspecific cell type or subset of cell types, and cannot, under normalcircumstances, differentiate into a different cell type or revert to aless differentiated cell type. “De-differentiation” refers to theprocess by which a cell reverts to a less specialized (or committed)position within the lineage of a cell. As used herein, the “lineage” ofa cell defines the heredity of the cell, i.e., which cells it came fromand what cells it can give rise to. The lineage of a cell places thecell within a hereditary scheme of development and differentiation.

In a broad sense, a “progenitor cell” is a cell that has the capacity tocreate progeny that are more differentiated than itself, and yet retainsthe capacity to replenish the pool of progenitors. By that definition,stem cells themselves are also progenitor cells, as are the moreimmediate precursors to terminally differentiated cells. When referringto the cells of the present invention, as described in more detailbelow, this broad definition of progenitor cell may be used. In anarrower sense, a progenitor cell is often defined as a cell that isintermediate in the differentiation pathway, i.e., it arises from a stemcell and is intermediate in the production of a mature cell type orsubset of cell types. This type of progenitor cell is generally not ableto self-renew. Accordingly, if this type of cell is referred to herein,it will be referred to as a “non-renewing progenitor cell” or as an“intermediate progenitor or precursor cell.”

Generally, a “trophic factor” is defined as a substance that promotessurvival, growth, proliferation, and/or maturation of a cell, orstimulates increased activity of a cell.

The term “standard growth conditions,” as used herein refers toculturing of cells at 37° C., in a standard atmosphere comprising 5% CO₂and relative humidity maintained at about 100%. While the foregoingconditions are useful for culturing, it is to be understood that suchconditions are capable of being varied by the skilled artisan who willappreciate the options available in the art for culturing cells.

The term “isolate” as used herein generally refers to a cell, which hasbeen separated from its natural environment. This term includes grossphysical separation from its natural environment, e.g., removal from thedonor animal. In preferred embodiments, an isolated cell is not presentin a tissue, i.e., the cell is separated or dissociated from theneighboring cells with which it is normally in contact. Preferably,cells are administered as a cell suspension. As used herein, the phrase“cell suspension” includes cells which are in contact with a medium andwhich have been dissociated, e.g., by subjecting a piece of tissue togentle trituration.

As used herein, the term peripheral blood mononuclear cell (“PBMC”)encompasses any blood cell having a round nucleus. Exemplary peripheralblood mononuclear cells include but are not limited to lymphocytes,monocytes, and macrophages.

II. Methods of Detecting Allogeneic Therapeutic Cells in a Patient BloodSample

This application provides for methods of detecting allogeneictherapeutic cells in a patient's blood sample after the cells have beenadministered to the patient. The methods involve the steps of (a)providing a blood sample from a patient that has been treated with theallogeneic therapeutic cells; (b) analyzing the sample using an assaymethod to detect one or more markers positive for patient peripheralblood mononuclear cells (“PBMC”) (e.g. any blood cell having a roundnucleus) and/or one or more markers positive for therapeutics cells; and(c) distinguishing between the human peripheral blood mononuclear cellsand one or more markers positive for allogeneic therapeutic cells thatare not expressed by the peripheral blood mononuclear cells. To be ableto distinguish between the markers for patient peripheral bloodmononuclear cells and one or more markers positive for therapeuticscells that are not expressed by the peripheral blood mononuclear cells,one needs to also first identify these markers. Thus, the methods mayfurther include the step of assaying for these markers. The methods ofthe invention allow detection of such cells without being limited by thekaryotype of the allogeneic therapeutic cell (i.e. XX vs. XY) and thegender of the patient. Thus, the methods are not constrained by gendernor are they limited to only identifying male allogeneic therapeuticcells (XX) in a female patient.

In one embodiment, the invention provides methods for detecting oridentifying human umbilical cord tissue-derived cells in a blood sample.The methods involve the steps of: (a) providing a blood sample from apatient that has been treated with human umbilical cord tissue-derivedcells; (b) analyzing the sample using an assay method to detect one ormore markers positive for human peripheral blood mononuclear cells andone or more markers positive for human umbilical cord tissue-derivedcells; and (c) distinguishing between the human peripheral bloodmononuclear cells and the human umbilical cord tissue-derived cells. Toable to distinguish, one also needs to assay for the unique markerprofile to be able to select the distinguishing unique marker profile. Afurther step of quantifying the amount of hUTC may also be employed. Inone embodiment, the markers shown in Example 3 may be used.

In another embodiment, the invention describes methods to distinguishand/or measure an hUTC cell therapy product following intravenousadministration in humans. The methods identify molecular markers thatare expressed at substantially higher levels in hUTC as compared tocells normally present in blood, i.e., peripheral blood mononuclearcells (“PBMC”). Since the methods rely on a unique marker profile forthe identification of hUTC, they allow detection of such cells withoutbeing limited by karyotype of the hUTC(XX vs. XY) and the gender of thepatient. They are not constrained by gender nor are they limited to onlymale hUTC. Thus, these methods permit analysis of both male and femalehUTC in both male and female subjects, in any combination.

The methods of the invention are suitable to distinguish allogeneictherapeutic cells in the blood of a patient from a variety of sources.In particular, the methods of the invention are suitable for detecting asmall number of hUTC in the presence of a large excess of a recipient'sblood. This method is applicable for any member of the mammalian systemand is not restricted to that derived from humans. For example, themethods of the invention may be used to distinguish allogeneictherapeutic cells such as e.g. hUTC from PBMC in a human. Alternatively,the methods may be used to distinguish allogeneic therapeutic cells suchas e.g. hUTC from PBMC in a non-human primate, mouse, rat, hamster,guinea pig, dog, or pig.

These methods help differentiate hUTC and other allogeneic therapeuticcells from the PBMC and may help monitor pharmacokinetics in subjectswho receive a cell therapy product.

The methods of the invention are suitable for detection of allogeneictherapeutic cells (such as e.g. hUTC) in blood samples from patientshaving been treated with the cells. Thus, the methods of the inventionare suitable for detection of allogeneic therapeutic cells (such as e.g.hUTC) that may have been previously administered. The methods of theinvention may be suitable for the detection of any cell of therapeuticinterest such as e.g. human cord blood-derived cells, placenta-derivedcells, mesenchymal stem cells and mesenchymal stem cell-derived cells,hUTC, cardiomyocytes etc. or any targeted cells expressing a specificprotein or proteins of interest. Other suitable cells for use in thesemethods include liver cells, pancreatic islet cells, fibroblasts andinsoluble collagenous bone matrix-derived (ICBM) cells.

The methods may also be suitable for detection of two or more kinds ofallogeneic therapeutic cells (such as e.g. hUTC) in blood samples frompatients having been treated with the cells. For example, the methodsmay be used to detect both human umbilical cord tissue-derived cells andplacenta-derived cells in a blood sample. Alternatively, the methods canbe used to detect human umbilical cord tissue-derived cells and dermalfibroblasts in a blood sample. The methods may also be used to detecthuman umbilical cord tissue-derived cells, placenta-derived cells, anddermal fibroblasts in a blood sample.

A. Identification of a Distinguishing Unique Marker Profile

The methods of the invention include the step of identifying adistinguishing unique marker profile based on the presence of thetherapeutic cell (such as e.g. a human umbilical cord tissue-derivedcells) in a patient blood sample containing PBMC.

To derive this unique marker profile, the expression of genes andpresence of cell surface markers needs to be compared between the cells(e.g. human umbilical cord tissue-derived cells) and PBMC. Multipletechnologies including protein-based technologies such as ELISA andimmune-histochemistry or nucleic acid based technologies such as in situhybridization may be used to detect the presence of hUTC in blood.Alternatively, PCR technology may be also used. As outlined in theExamples below, the expression level of various genes in PBMC and/or theallogeneic therapeutic cells may also be obtained from publiclyavailable sources.

Based on the expression level of these various genes, certain uniquemarker genes may be identified. In one embodiment, particularly suitablefor rapid screening, the marker is a cell surface marker.

As illustrated in the examples below, molecular markers on hUTC may beidentified by comparing the expression profile of these cells to that ofhuman peripheral blood mononuclear cells (PBMC). By comparing expressionprofiles of hUTC and PBMC markers that are expressed at substantiallyhigher levels in hUTC compared to cells normally present in thecirculation and vice versa are thus identified for each patient. Themarkers identified include CD45, CD13, and CD10. As both hUTC andcirculating cells can show dynamic expression levels, the method relieson multiple markers that can distinguish hUTCs from cells in therecipient's circulation.

The unique marker profile may include one or more markers positive forthe allogeneic cells, the presence of which is being assayed, and one ormore markers positive for the peripheral blood mononuclear cells. Thus,when assaying for human umbilical cord tissue-derived cells, the markerprofile may include one or more markers positive for hUTC and one ormore markers positive for peripheral blood mononuclear cells.Alternatively, the allogeneic cells, the presence of which is beingassayed, may be identified using only one or more unique markers for theallogeneic cells sufficient to distinguish these cells from theperipheral blood mononuclear cells.

In one embodiment, particularly useful for identifying hUTC in PBMC, theunique marker profile includes one or more of CD10, CD13, NRP1, CD45,LAMP1, DKK3, NRP1, or LAMB1. In one embodiment, the unique markerprofile comprises at least one or more marker characteristic (e.g.positive) for the hUTC and at least one marker characteristic (e.g.positive) for PBMC (such as e.g. CD45).

In another embodiment, also particularly useful for identifying hUTC inPBMC, the unique marker profile includes one or more unique markers thatare positive for the hUTC and that are sufficient to distinguish hUTCfrom the peripheral blood mononuclear cells. These one or more uniquemarkers may include one or more of CD10, CD13, NRP1, LAMP1, DKK3, NRP1,or LAMB1.

Those of skill will recognize that the choice of which marker to use mayvary depending on which technique is used. For example, using amicroarray analysis (such as e.g. Affymetrix GeneChip® HT HG-U133+ PMarray), ANPEP (CD13), LAMP1 and LAMB1 may be insufficient to distinguishwhile one or more of the others markers may be sufficient todistinguish. Similarly, using RT-PCT, LAMP1 may not be suitable as partof the unique marker profile that distinguishes hUTC from PBMC, whileone or more of the other markers may be sufficient. In one embodiment,the unique marker profile used for screening for hUTC by cell surfaceflow cytometry does not include DKK3 and LAMB1. In another embodiment,the unique marker profile for intracellular cytometry includes LAMP1 andDKK3.

Exemplary suitable markers for identifying hUTC in a PBMC sample, usingselected techniques are shown below.

Markers only Markers only positive for positive for PBMC Technique hUTCusing technique using technique Microarray analysis MME (CD10), NRP1,DKK3 PTPRC (CD45) RT-PCR MME (CD10), NRP1, DKK3, PTPRC (CD45) LAMB1 Flowcytometry MME (CD10), ANPEP PTPRC (CD45) (surface) (CD13) Flow cytometryLAMP1 (intracellular)

Thus, in one embodiment, the unique marker profile includes CD10 as theone or more markers positive hUTC and CD45 as the one or more markerspositive for PBMC. In another embodiment, the unique marker profileincludes CD10 and/or CD13 as the one or more markers positive hUTC andCD45 as the one or more markers positive for PBMC.

In another embodiment, using flow cytometry as the assay method, theunique marker profile for identifying hUTC in PBMC includes CD45 as theone or more markers positive for peripheral blood mononuclear cell andCD10 as one or more markers positive for human umbilical cordtissue-derived cells.

In another embodiment of the invention, the maker profile to distinguishhUTC and PBMC comprises CD45, CD10, and CD13 using flow cytometry,wherein CD10 and CD13 are the one or more markers positive for hUTC andwherein CD45 is the one or more markers positive for PBMC.

Exemplary suitable markers for the identifying selected therapeuticcells in a PBMC sample are shown below:

Marker for Type of cell Marker for cell type host PBMC Human umbilicalcord-blood CD10, CD13, CD107a CD45 derived Placenta derived Renin Heatshock 27 kd Mesenchymal stem cell derived IL26, a-glucosidase CD45 LiverFOXa2, HNFa1 CD45 Pancreatic islet cells G6PC2, INSULIN CD45 FibroblastSDF-1, GCP-2 CD45 Human umbilical cord-blood IL8, GCP-2 CD45 derived andplacenta-derived cells and dermal fibroblasts Human umbilical cord-bloodIL6, MCP-1 CD45 derived and placenta-derived cells Insoluble collagenousbone Integrin a-10; cardiac CD45 matrix (ICBM) ankyrin repeat proteinCardiomyocytes FoxA2, GATA4, MIR20 CD45

The unique marker profile is obtainable using the following proceduresas well the methodology described in the examples. The proceduresdescribe below may encompass both obtaining the unique marker profileand verifying its accuracy. While the step of verification may encompassanalysis of a sample, the step may be necessary to determine theaccuracy and testing limits of the specific procedure by which theunique marker gene or protein will be detected. In this embodiment, flowcytometry may be used to identify the markers.

Obtaining the Unique Marker Profile.

The gene expression profile of hUTC, sourced from multiple sources, wasgenerated using an Affymetrix GeneChip® HT HG-U133+ PM. The geneexpression profiles of human and rat PBMC and other types of blood cellswere obtained from publicly available databases. From the results of thegene expression profiles, a set of markers that are highly expressed inhUTC and a set of markers that are highly expressed in human PBMC can beidentified. Sixty-one (61) genes were thus identified, a subset of whichwere studied in more detail. From this list of 61 genes, CD45 wasidentified as a positive marker for hPBMC and CD10 and CD13 as twopositive markers for hUTC.

Verifying the Testing Methodology for the Marker Profile.

Since the number of hUTC in a sample of a subject's blood is anticipatedto be low, it was determined whether a small number of hUTC in thepresence of a large excess of human PBMC using the above-identifiedmarkers could be detected and quantified. For this purpose, knownquantities of hUTC were spiked into approximately one million PBMC in 1ml of human serum. Subsequently, an aliquot of each sample was analyzedby flow cytometry for CD45 as a positive marker for hPBMC and CD10 orCD13 as positive markers for hUTC. The cells were harvested bycentrifugation, washed once with PBS and then fixed and permeabilized.Aliquots of cells were then incubated with: (1) FITC-labeled anti-CD45(Abcam, Cat #27287); (2) mouse monoclonal to CD10 (Abcam, Cat #34175)followed by FITC-labeled goat anti-mouse Ab (Abcam, Cat #6785); and (3)mouse monoclonal to CD13 (Abcam, Cat #7417) followed by FITC-labeledgoat anti-mouse Ab (Abcam, Cat #6785). Propidium iodide (PI) wasincluded in each sample to monitor viability as only live cells weregated for further analysis. Using this procedure, it was confirmed thattesting for a marker profile comprising CD10, CD13, and CD45 using flowcytometry is successful to identify hUTC and PBMC.

In one embodiment, the unique marker profile may be provided as part ofa kit containing the materials for testing for the unique markerprofile.

The step of identifying the unique marker profile may also include thestep of verifying its correctness and possible validation of the uniquemarker profile in compliance with relevant regulations.

B. Obtaining a Patient Sample

The step of obtaining a patient sample includes the step of taking ablood sample from the patient. As mentioned above, in one embodiment,the patient may be a human, non-human primate, mouse, rat, hamster,guinea pig, dog, or pig. The blood sample may be further purified orconcentrated to isolate the cells. For example, in one embodiment, theplasma may be removed from the blood. The plasma may be removed usingstandard techniques in the art including centrifugation.

The patient sample (e.g. fraction) used for further analysis comprisesthe allogeneic therapeutic cells of interest and the peripheral bloodmononuclear cells from the patient. In one embodiment, the patientsample comprises a human umbilical cord tissue-derived/peripheral bloodmononuclear cell fraction.

In one embodiment, the step of obtaining a patient sample furtherincludes isolating the human umbilical cord tissue-derived/peripheralblood mononuclear cell fraction from the blood sample. This embodimentmay further include the step of removing plasma.

In an alternate embodiment, the step of obtaining a patient sampleincludes use of Ficoll extraction.

C. Analysis of the Patient Sample

Generally, the step of analysis comprises taking the patient bloodsample and testing it for the presence of the distinguishing uniquemarker profile for allogeneic therapeutic cells (e.g. hUTC). The step ofanalysis may also include determining the presence of PBMC based on aunique marker profile as determined above. For instance, the step ofanalyzing the patient sample includes use of an assay method to test forone or more markers positive for the allogeneic therapeutic cells andone or more markers positive for peripheral blood mononuclear cells.Thus, the step of analysis includes detecting the presence of cellsbased on the unique marker profile assayed above. Alternatively, thestep of analyzing the patient sample includes use of an assay method totest for one or more unique markers positive for the allogeneictherapeutic cell sufficient to distinguish these cells from theperipheral blood mononuclear cells.

In one embodiment, the step of analysis includes detecting one or moremarkers positive for peripheral blood mononuclear cells and one or moremarkers positive for human umbilical cord tissue-derived cells in thepatient blood sample. In another embodiment, the step of analysisincludes detecting one or more unique markets positive for hUTCsufficient to distinguish these cells from PBMC in the patient bloodsample. The patient blood sample may be a human umbilical cordtissue-derived/peripheral blood mononuclear cell fraction.

The step of analysis may be carried out using a variety of standardlaboratory techniques. In one embodiment, the step of analyzing mayutilize flow cytometry, ELISA, immunohistochemistry, nucleic aciddetection, PCR, or combinations thereof.

Multiple technologies including protein-based technologies such as ELISAand immune-histochemistry or nucleic acid based technologies such as insitu hybridization may be used to detect the presence of hUTC in blood.Alternatively, PCR technology may be used to facilitate the relativequantification of hUTC in a blood sample.

In one preferred embodiment of the invention, the methods of theinvention are used to distinguish human hUTC from human PBMC. In oneembodiment, the unique marker profile comprises one or more of CD10,CD13, NRP1, CD45, LAMP1, DKK3, NRP1, or LAMB1 and the step of analyzingmay utilize flow cytometry, ELISA, immunohistochemistry, nucleic aciddetection, PCR, or combinations thereof. In another embodiment, step ofanalysis includes flow cytometry to test for CD10 and/or CD13 as apositive marker for human umbilical cord tissue-derived cells and CD45as a positive marker for human peripheral blood mononuclear cells. Inanother embodiment, the methods of the invention are used to distinguishhUTC from human PBMC based on the presence of one or more uniquepositive markers (such as e.g. CD10, CD13, NRP1, LAMP1, DKK3, NRP1, orLAMB1) sufficient to distinguish these cells from the PBMC.

D. Optional Enrichment Steps

The methods of the invention may further include one or more enrichmentsteps. The term “enrichment” as used herein refers to the process ofsubstantially increasing the ratio of target bioentities (e.g.,allogeneic therapeutic cells (such as e.g. hUTC)) to non-targetmaterials (e.g. PBMC) in the processed analytical sample compared to theratio in the original biological sample.

In particular, if more sensitivity is required for detection of theallogeneic therapeutic cells (e.g. hUTC), an enrichment step may benecessary prior to analyzing the patient sample. For example, anenrichment step may be necessary where there are so few allogeneictherapeutic cells (e.g. hUTC) present relative to the number of bloodcells or that the amount of allogeneic therapeutic cells falls below thelimits of detection of the laboratory technique. Using enrichment, themethods of the invention can identify and quantify allogeneictherapeutic cells, even if present in very low concentration in thepatient's blood and provide a mechanism to facilitate quantification ofcells present in any given sample.

A variety of standard techniques may be used for the one or moreenrichment steps. These techniques include, but are not limited to, theuse of selective medium for enrichment and depletion of specific celltypes, selective adhesions method, physical and biological methods ofcell separation, density gradient electrophoresis, centrifugation andthe like. Exemplary enrichment techniques are immunoaffinity column,immunoaffinity beads, centrifugation through a sucrose or Ficollgradient.

Thus, depending on the number of hUTC/ml of blood and the number ofhUTC/million PBMC, it may be necessary to isolate or enrich the hUTCfraction prior to analysis. Anti-CD45 antibody, conjugated to magneticbeads may be used for this purpose (see discussion below).

Cell Separation with Magnetic Beads

The enrichment step may include cell separation with magneticparticles/beads. Magnetic particles are well known in the art, as istheir use in immune and other bio-specific affinity reactions.Generally, any material that facilitates magnetic or gravitationalseparation may be employed for this purpose. Exemplary enrichmentprocedures using magnetic beads are described in U.S. Pat. No. 7,863,012and U.S. Published Application No. 2011/0147180, which are incorporatedby reference.

Magnetic particles can be classified based on size: large (1.5 to about50 microns); small (about 0.7-1.5 microns); or colloidal (<200 nm),which are also referred to as nanoparticles. The third, which are alsoknown as ferrofluids or ferrofluid-like materials and have many of theproperties of classical ferrofluids, are sometimes referred to herein ascolloidal, superparamagnetic particles.

Small magnetic particles of the type described above are quite useful inanalyses involving bio-specific affinity reactions, as they areconveniently coated with biofunctional polymers (e.g., proteins),provide very high surface areas, and give reasonable reaction kinetics.Magnetic particles ranging from about 0.7-1.5 microns have beendescribed in the patent literature, including, by way of example, U.S.Pat. Nos. 3,970,518; 4,018,886; 4,230,685; 4,267,234; 4,452,773;4,554,088; and 4,659,678. Certain of these particles are disclosed to beuseful solid supports for immunological reagents.

The efficiency with which magnetic separations can be done and therecovery and purity of magnetically labeled cells will depend on manyfactors. These include: number of cells being separated, receptor orepitope density of such cells, magnetic load per cell, non-specificbinding (NSB) of the magnetic material, carry-over of entrappednon-target cells, technique employed, nature of the vessel, nature ofthe vessel surface, viscosity of the medium, and magnetic separationdevice employed. If the level of non-specific binding of a system issubstantially constant, as is usually the case, then as the targetpopulation decreases so will the purity.

As an example, a system with 0.8% NSB that recovers 80% of a population,which is at 0.25% in the original mixture, will have a purity of 25%.Whereas, if the initial population was at 0.01% (one target cell in 10⁶bystander cells), and the NSB were 0.001%, then the purity would be 8%.Hence, a high the purity of the target material in the specimen mixtureresults in a more specific and effective collection of the targetmaterial. Extremely low non-specific binding is required or advantageousto facilitate detection and analysis of rare cells, such as epithelialderived tumor cells present in the circulation.

The smaller the population of a targeted cell (such as e.g. hUTC), themore difficult it will be to magnetically label and to recover.Furthermore, labeling and recovery will markedly depend on the nature ofmagnetic particle employed. For example, when cells are incubated withlarge magnetic particles, such as Dynal® magnetic beads (Invitrogen),cells are labeled through collisions created by mixing of the system, asthe beads are too large to diffuse effectively. Thus, if a cell werepresent in a population at a frequency of 1 cell per ml of blood or evenless, then the probability of labeling target cells will be related tothe number of magnetic particles added to the system and the length oftime of mixing. Since mixing of cells with such particles forsubstantial periods would be deleterious, it becomes necessary toincrease particle concentration as much as possible. There is, however,a limit to the quantity of magnetic particle that can be added, as onecan substitute a rare cell mixed in with other blood cells for a rarecell mixed in with large quantities of magnetic particles uponseparation. The latter condition does not markedly improve the abilityto enumerate the cells of interest or to examine them.

In one embodiment, the magnetic particles for use in carrying out theenrichment step behave as colloids. Such particles are characterized bytheir sub-micron particle size, which is generally less than about 200nm (0.20 microns), and their stability to gravitational separation fromsolution for extended periods. In addition to the many other advantages,this size range makes them essentially invisible to analyticaltechniques commonly applied to cell analysis. Particles within the rangeof about 90-150 nm and having between about 70-90% magnetic mass arecontemplated for use in the present invention. Suitable magneticparticles are composed of a crystalline core of superparamagneticmaterial surrounded by molecules, which are bonded, e.g., physicallyabsorbed, or covalently attached to the magnetic core and which conferstabilizing colloidal properties. The coating material should preferablybe applied in an amount effective to prevent non-specific interactionsbetween biological macromolecules found in the sample and the magneticcores. Such biological macromolecules may include carbohydrates such assialic acid residues on the surface of non-target cells, lectins,glycoproteins, and other membrane components. In addition, the materialshould contain as much magnetic mass per nanoparticle as possible. Thesize of the magnetic crystals comprising the core is sufficiently smallthat they do not contain a complete magnetic domain. The size of thenanoparticles is sufficiently small such that their Brownian energyexceeds their magnetic moment. Consequently, North Pole, South Polealignment and subsequent mutual attraction/repulsion of these colloidalmagnetic particles does not appear to occur even in moderately strongmagnetic fields, contributing to their solution stability. Finally, themagnetic particles should be separable in high magnetic gradientexternal field separators. That characteristic facilitates samplehandling and provides economic advantages over the more complicatedinternal gradient columns loaded with ferromagnetic beads or steel wool.Magnetic particles having the above-described properties can be preparedby modification of base materials described in U.S. Pat. Nos. 4,795,698;5,597,531, and 5,698,271, each incorporated by reference herein.

Since small nanoparticles (30-70 nm) will diffuse more readily, theywill preferentially label cells compared with their larger counterparts.When very high gradients are used, such as in internal gradient columns,the performance of these materials, regardless of size, makes littledifference. On the other hand, when using external gradients, orgradients of lesser magnitude than can be generated on micro bead orsteel wool columns, the occupancy of small nanoparticles on cells has asignificant effect. This was conclusively shown to be the case byfractionating DC nanoparticles and studying the effects on recovery.Based on these studies and other optimization experiments, means forfractionating nanoparticles magnetically or on columns was establishedwhere base coated magnetic particles could be prepared that were devoidof excessively small or large nanoparticles. For example, base coatedparticles of mean diameter 100 nm can be produced which contain at besttrace amounts of material smaller than 80 nm or over 130 nm. Similarly,material of about 120 nm can be made with no appreciable materialsmaller than 90-95 nm and over 160 nm. Such materials performedoptimally with regard to recovery and could be made sub-optimal by theinclusion of 60-70 nm nanoparticles. One preferred particle size rangefor use in practicing this invention is 90-150 nm for base coatedmagnetic particles, e.g., BSA-coated magnetite.

Based on the foregoing, high gradient magnetic separation with anexternal field device employing highly magnetic, low non-specificbinding, colloidal magnetic particles is the method of choice forseparating a cell subset of interest from a mixed population ofeukaryotic cells (such as e.g. allogeneic therapeutic cells (e.g. hUTC)in peripheral blood mononuclear cells), particularly if the subset ofinterest comprises but a small fraction of the entire population. Suchmaterials, because of their diffusive properties, readily find andmagnetically label rare events, such as tumor cells in blood. In oneembodiment, for magnetic separations for allogeneic therapeutic (e.g.hUTC) cell analysis to be successful, the magnetic particles must bespecific for epitopes that are not present on the allogeneic therapeuticcells (e.g. hUTC).

An enrichment step by use of magnetic beads includes using magneticnanoparticles (such as those described above) which are labeled with amonoclonal antibody. These monoclonal antibodies may be antibodiesidentifying peripheral blood mononuclear cells but not allogeneictherapeutic cells. The monoclonal antibody attached to the nanoparticlesbind to the peripheral blood mononuclear cells which may then beseparated using magnetic means. Typically, such separation is achievedvia the use of a high magnetic gradient external field separators. Anexemplary suitable technique to separate by magnetic means is describedin U.S. Pat. No. 6,365,362, the disclosure which is incorporated herein.In one embodiment, an Anti-CD45 antibody conjugated to a magneticparticle is used. Alternatively, the enrichment step includes use ofAnti-CD4 and anti-CD8 antibodies, to remove T-cells.

In another embodiment, the enrichment step includes monoclonalantibodies identifying the allogeneic therapeutic cells. In a furtherembodiment, the allogeneic therapeutic cells are hUTC and the antibodiesinclude one or more of anti-CD10 or anti-CD13 antibodies.

In one embodiment, the methods of the invention include use of a CellSearch System (Veridex, LLC (Raritan, N.J.)), which has been optimizedfor the detection of hUTC and PBMC.

As the approximate amount of allogeneic therapeutic cells (such as e.g.hUTC) in a patient sample may be unknown, the methods of the inventionmay also include the step of preserving a sufficient patient sample torun an additional analysis. This may be particularly useful whereinitial analysis might not be able to detect the allogeneic therapeuticcells (such as e.g. hUTC) due to allogeneic therapeutic cells beingpresent in an amount below the detection limit of the assay techniqueused. Thus, in one embodiment, the method further includes an additionalanalysis of a patient sample if no allogeneic therapeutic cells areinitially detected (such as e.g. hUTC). This additional analysis alsoincludes one or more enrichment steps such as those discussed above.

E. Determining Presence of Allogeneic Therapeutic Cells and PeripheralBlood Mononuclear Cells in the Patient's Blood Sample

The methods of the invention further comprise determining the presenceof allogeneic therapeutic cells in a sample of peripheral bloodmononuclear cells. The step of determining the presence of said cellsincludes comparing the results of the analysis of the sample to theunique marker, which was assayed for above, wherein the presence ofunique markers for allogeneic therapeutic cells indicates the presenceof the allogeneic therapeutic cells in the sample. The step ofdetermining may also include comparing the result to a unique marker forthe PBMC as identified above. Thus, the determination step includescomparing the results for the one or more markers positive for theallogeneic therapeutic cell (e.g. hUTC) and the one or more markersnegative for PBMC to the marker profile above.

In another embodiment, the method of the invention comprises determiningthe presence of human umbilical cord tissue-derived in a sample ofperipheral blood mononuclear cells (such as e.g. human or rodent). Thedetermination step includes comparing the results of the analysis of thesample to the unique marker, which was assayed for above, wherein thepresence of unique markers for the allogeneic therapeutic cellsindicates the presence of the hUTC cells in the sample. Thedetermination step may also include comparing the result to a uniquemarker for the PBMC as identified above. The determination step mayinclude one or more markers positive for the hUTC and one or moremarkers positive for PBMC as discussed above.

The determination step also includes distinguishing between the humanperipheral blood mononuclear cells and human umbilical cordtissue-derived cells based on the detection of one or more markerspositive for human peripheral blood mononuclear cells and one or moremarkers positive for human umbilical cord tissue-derived cells. Thedetermination step may also include differentiating between humanumbilical cord tissue-derived cells administered to the patient andhuman peripheral blood mononuclear cells from the patient.

The step of determining the presence of the cells may also includequantifying the cells. In one embodiment, the determination step for thepresence of hUTC includes quantifying the number of hUTC in the sample.

The step of determining the presence may further include selecting oneor more human umbilical cord tissue-derived cells based on the presenceof the unique marker profile (such as e.g. one or more markers positivefor hUTC).

The determination step may also include detecting the presence of thehuman peripheral blood mononuclear cells and human umbilical cordtissue-derived cells based on the detection of CD45 as a marker positivefor human peripheral blood mononuclear cells and CD10 or CD13 as amarker positive for human umbilical cord tissue-derived cells.

In alternate embodiments, the methods described herein may be suitableto distinguish allogeneic therapeutic cells from rat PBMC, which may beparticularly useful for monitoring in rat disease models.

In one embodiment, the present invention describes a method for theidentification of hUTC in blood. The method involves the followingsteps: (a) providing a blood sample from a patient that has been treatedwith human umbilical cord tissue-derived cells; (b) analyzing the sampleusing an assay method to detect one or more markers positive for humanperipheral blood mononuclear cells and one or more markers positive forhuman umbilical cord tissue-derived cells; and (c) distinguishingbetween the human peripheral blood mononuclear cells and one or moremarkers positive for human umbilical cord tissue-derived cells. Afurther step of quantifying the amount of hUTC may also be employed. Inone embodiment, one or more of the markers shown in Example 3 are used.

In another embodiment, the methods of the invention are suitable foridentifying about 1,700 or more hUTC/ml in the presence of 1 millionhuman PBMC using flow cytometry without relying on an enrichment step.

III. Kits and Systems for Testing for Allogeneic Therapeutic Cells in aBlood Sample

Another embodiment of the invention is a kit for testing for thepresence of allogeneic therapeutic cells such as hUTC in a blood sampleusing the methods of the invention.

The kits may comprise the distinguishing unique marker profile andsuitable components for identifying the distinguishing unique markerprofile according to one or more analytical procedures. For example, akit suitable for distinguishing a unique marker profile by using RT-PCRwould include PCR primers suitable for amplifying the unique markerprofile genes. Similarly, a kit suitable for distinguishing a uniquemarker profile by an antibody-linked assay would include the antibodiesthat bind to the markers.

In one embodiment, the kit is designed to detect hUTC in a population ofPBMC. In another embodiment, the kit comprises the unique marker profileCD10, CD13 and CD45 and other suitable components for identifying CD10,CD13 and CD45 using one or more of flow cytometry, ELISA,immunohistochemistry, nucleic acid detection, PCR or combinationsthereof.

Yet another embodiment is a system for detecting allogeneic therapeuticcells (such as e.g. hUTC) in a population of PBMC. The system includesmaterials necessary for carrying out the methods of the invention.

IV. Human Umbilical Cord Tissue-Derived Cells

While it is contemplated that the methods of the invention may be usedto distinguish therapeutic (e.g. progenitor/stem cells) from host cells,in a preferred embodiment, the cells may be human umbilical cordtissue-derived-cells (“hUTC” or “UTC”). Useful human umbilical cordtissue-derived cells are isolated from human umbilical cord tissuesubstantially free of blood, are capable of self-renewal and expansionin culture, and have the potential to differentiate. The UTC and UTCpopulations suitable for identification by the methods of the inventionare described in detail in detailed herein below as well as U.S. Pat.Nos. 7,510,873; 7,524,489; and U.S. Pub. App. No. 2005/0058631.

A. Isolation and Growth of Umbilical Cord Tissue-Derived Cells

According to the methods described herein, a mammalian umbilical cord isrecovered upon or shortly after termination of either a full-term or apre-term pregnancy, e.g., after expulsion of after birth. The postpartumtissue may be transported from the birth site to a laboratory in asterile container such as a flask, beaker, culture dish, or bag. Thecontainer may have a solution or medium, including but not limited to asalt solution, such as Dulbecco's Modified Eagle's Medium (DMEM) (alsoknown as Dulbecco's Minimal Essential Medium) or phosphate bufferedsaline (PBS), or any solution used for transportation of organs used fortransplantation, such as University of Wisconsin solution orperfluorochemical solution. One or more antibiotic and/or antimycoticagents, such as but not limited to penicillin, streptomycin,amphotericin B, gentamicin, and nystatin, may be added to the medium orbuffer. The postpartum tissue may be rinsed with an anticoagulantsolution such as heparin-containing solution. It is preferable to keepthe tissue at about 4-10° C. prior to extraction of UTC. It is even morepreferable that the tissue not be frozen prior to extraction of UTC.

Isolation of UTC preferably occurs in an aseptic environment. Theumbilical cord may be separated from the placenta by means known in theart. Blood and debris are preferably removed from the postpartum tissueprior to isolation of UTC. For example, the postpartum tissue may bewashed with buffer solution, including but not limited to phosphatebuffered saline. The wash buffer also may comprise one or moreantimycotic and/or antibiotic agents, including but not limited topenicillin, streptomycin, amphotericin B, gentamicin, and nystatin.

Postpartum tissue comprising an umbilical cord or a fragment or sectionthereof is disaggregated by mechanical force (mincing or shear forces).In a presently preferred embodiment, the isolation procedure alsoutilizes an enzymatic digestion process. Many enzymes are known in theart to be useful for the isolation of individual cells from complextissue matrices to facilitate growth in culture. Digestion enzymes rangefrom weakly digestive (e.g. deoxyribonucleases and the neutral protease,dispase) to strongly digestive (e.g. papain and trypsin), and areavailable commercially. A non-exhaustive list of enzymes compatibleherewith includes mucolytic enzyme activities, metalloproteases, neutralproteases, serine proteases (such as trypsin, chymotrypsin, orelastase), and deoxyribonucleases. Presently preferred are enzymeactivities selected from metalloproteases, neutral proteases andmucolytic activities. For example, collagenases are known to be usefulfor isolating various cells from tissues. Deoxyribonucleases can digestsingle-stranded DNA and can minimize cell clumping during isolation.Preferred methods involve enzymatic treatment with e.g. collagenase anddispase, or collagenase, dispase, and hyaluronidase. In certainembodiments, a mixture of collagenase and the neutral protease dispaseare used in the dissociating step. More specific embodiments employdigestion in the presence of at least one collagenase from Clostridiumhistolyticum, and either of the protease activities, dispase, andthermolysin. Still other embodiments employ digestion with bothcollagenase and dispase enzyme activities. Also utilized are methodsthat include digestion with a hyaluronidase activity in addition tocollagenase and dispase activities. The skilled artisan will appreciatethat many such enzyme treatments are known in the art for isolatingcells from various tissue sources. For example, the enzyme blends fortissue disassociation sold under the trade name LIBERASE (Roche,Indianapolis, Ind.) are suitable for use in the instant methods. Othersources of enzymes are known, and the skilled artisan may also obtainsuch enzymes directly from their natural sources. The skilled artisan isalso well-equipped to assess new or additional enzymes or enzymecombinations for their utility in isolating the cells of the invention.Preferred enzyme treatments are 0.5, 1, 1.5, or 2 hours long or longer.In other preferred embodiments, the tissue is incubated at 37° C. duringthe enzyme treatment of the dissociation step.

In some embodiments of the invention, postpartum tissue is separatedinto sections comprising various aspects of the tissue, such asneonatal, neonatal/maternal, and maternal aspects of the placenta, forinstance. The separated sections then are dissociated by mechanicaland/or enzymatic dissociation according to the methods described herein.Cells of neonatal or maternal lineage may be identified by any meansknown in the art, e.g. by karyotype analysis or in situ hybridizationfor a Y chromosome.

Isolated cells or umbilical cord tissue from which a UTC is derived maybe used to initiate, or seed, cell cultures. Isolated cells aretransferred to sterile tissue culture vessels either uncoated or coatedwith extracellular matrix or ligands such as laminin, collagen (native,denatured or cross-linked), gelatin, fibronectin, and otherextracellular matrix proteins. In addition to the culture mediadisclosed herein, a UTC may be cultured in any culture medium capable ofsustaining growth of the cell such as, but not limited to, DMEM (high orlow glucose), advanced DMEM, DMEM/MCDB 201, Eagle's basal medium, Ham'sF10 medium (F10), Ham's F-12 medium (F12), Iscove's modified Dulbecco'smedium, Mesenchymal Stem Cell Growth Medium (MSCGM), DMEM/F12, RPMI1640, and serum/media free medium sold under the trade nameCELL-GRO-FREE (Mediatch, Inc., Herndon, Va.). The culture medium may besupplemented with one or more components including, e.g., fetal bovineserum (FBS), preferably about 2-15% (v/v); equine serum (ES); humanserum (HS); beta-mercaptoethanol (BME or 2-ME), preferably about 0.001%(v/v); one or more growth factors, e.g., platelet-derived growth factor(PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF),vascular endothelial growth factor (VEGF), insulin-like growth factor-1(IGF-1), leukocyte inhibitory factor (LIF) and erythropoietin (EPO);amino acids, including L-valine; and one or more antibiotic and/orantimycotic agents to control microbial contamination, such aspenicillin G, streptomycin sulfate, amphotericin B, gentamicin, andnystatin, either alone or in combination. The culture medium maycomprise Growth Medium as defined in the Examples below.

The cells are seeded in culture vessels at a density to allow cellgrowth. In a preferred embodiment, the cells are cultured at about 0 toabout 5% by volume CO₂ in air. In some preferred embodiments, the cellsare cultured at about 2 to about 25% O₂ in air, preferably about 5 toabout 20% O₂ in air. The cells preferably are cultured at a temperatureof about 25 to about 40° C. and more preferably are cultured at 37° C.The cells are preferably cultured in an incubator. The medium in theculture vessel can be static or agitated, e.g., using a bioreactor. TheUTC is preferably grown under low oxidative stress (e.g., with additionof glutathione, Vitamin C, Catalase, Vitamin E, N-Acetylcysteine). “Lowoxidative stress” refers to conditions of no or minimal free radicaldamage to the cultured cells.

Methods for the selection of the most appropriate culture medium, mediumpreparation, and cell culture techniques are well known in the art andare described in a variety of sources, including Doyle et al., (eds.),1995, Cell & Tissue Culture: Laboratory Procedures, John Wiley & Sons,Chichester; and Ho and Wang (eds.), 1991, Animal Cell Bioreactors,Butterworth-Heinemann, Boston, which are incorporated herein byreference.

After culturing the isolated cells or tissue fragments for a sufficientperiod, a UTC will have grown out, either because of migration from thepostpartum tissue or cell division, or both. In some embodiments of theinvention, the UTC is passaged, or removed to a separate culture vesselcontaining fresh medium of the same or a different type as that usedinitially, where the population of cells can be mitotically expanded.The cells of the invention may be used at any point between passage 0and senescence. The cells preferably are passaged between about 3 andabout 25 times, more preferably are passaged about 4 to about 12 times,and preferably are passaged 10 or 11 times. Cloning and/or subcloningmay be performed to confirm that a clonal population of cells has beenisolated.

In certain embodiments, the different cell types present in postpartumtissue are fractionated into subpopulations from which the UTC can beisolated. Fractionation or selection may be accomplished using standardtechniques for cell separation including, but not limited to, enzymatictreatment to dissociate postpartum tissue into its component cells,followed by cloning and selection of specific cell types, including butnot limited to selection based on morphological and/or biochemicalmarkers; selective growth of desired cells (positive selection),selective destruction of unwanted cells (negative selection); separationbased upon differential cell agglutinability in the mixed populationsuch as, e.g., with soybean agglutinin; freeze-thaw procedures;differential adherence properties of the cells in the mixed population;filtration; conventional and zonal centrifugation; centrifugalelutriation (counter-streaming centrifugation); unit gravity separation;countercurrent distribution; electrophoresis; and fluorescence activatedcell sorting (FACS). For a review of clonal selection and cellseparation techniques, see Freshney, 1994, Culture of Animal Cells: AManual of Basic Techniques, 3rd Ed., Wiley-Liss, Inc., New York, whichis incorporated herein by reference.

The culture medium is changed as necessary, e.g., by carefullyaspirating the medium from the dish, e.g., with a pipette, andreplenishing with fresh medium. Incubation is continued until asufficient number or density of cells accumulates in the dish. Theoriginal explanted tissue sections may be removed and the remainingcells trypsinized using standard techniques or using a cell scraper.After trypsinization, the cells are collected, removed to fresh medium,and incubated as above. In some embodiments, the medium is changed atleast once at approximately 24 hours post-trypsinization to remove anyfloating cells. The cells remaining in culture are considered to be UTC.

The UTC may be cryopreserved. Accordingly, UTC for autologous transfer(for either the mother or child) may be derived from appropriatepostpartum tissues following the birth of a child, then cryopreserved soas to be available in the event they are later needed fortransplantation.

B. Characteristics of Umbilical Cord Tissue-Derived Cells

While hUTC may be distinguished from PBMC based on the presence of e.g.CD45, CD13, and CD10 and other markers discussed above and in theexamples below, hUTC possess a variety of other unique characteristics.

The UTC may be characterized, e.g., by growth characteristics (e.g.,population doubling capability, doubling time, passages to senescence),karyotype analysis (e.g., normal karyotype; maternal or neonatallineage), flow cytometry (e.g., FACS analysis), immunohistochemistryand/or immunocytochemistry (e.g., for detection of epitopes), geneexpression profiling (e.g., gene chip arrays; polymerase chain reaction(e.g., reverse transcriptase PCR, real time PCR, and conventional PCR)),protein arrays, protein secretion (e.g., by plasma clotting assay oranalysis of PDC-conditioned medium, e.g., by Enzyme Linked ImmunoSorbentAssay (ELISA)), mixed lymphocyte reaction (e.g., as measure ofstimulation of PBMCs), and/or other methods known in the art.

Examples of suitable UTC derived from umbilicus tissue were depositedwith the American Type Culture Collection (10801 University Boulevard,Manassas, Va. 20110) on Jun. 10, 2004, and assigned ATCC AccessionNumbers as follows: (1) strain designation UMB 022803 (P7) was assignedAccession No. PTA-6067; and (2) strain designation UMB 022803 (P17) wasassigned Accession No. PTA-6068.

In various embodiments, the UTC possesses one or more of the followinggrowth features: (1) they require L-valine for growth in culture; (2)they are capable of growth in atmospheres containing oxygen from about5% to at least about 20%; (3) they have the potential for at least about40 doublings in culture before reaching senescence; and (4) they attachand expand on a coated or uncoated tissue culture vessel, wherein thecoated tissue culture vessel comprises a coating of gelatin, laminin,collagen, polyornithine, vitronectin or fibronectin.

In certain embodiments, the UTC possesses a normal karyotype, which ismaintained as the cells are passaged. Methods for karyotyping areavailable and known to those of skill in the art.

In other embodiments, the UTC may be characterized by production ofcertain proteins, including: (1) production of at least one of tissuefactor, vimentin, and alpha-smooth muscle actin; and (2) production ofat least one of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, PD-L2 andHLA-A,B,C cell surface markers, as detected by flow cytometry. In otherembodiments, the UTC may be characterized by lack of production of atleast one of CD31, CD34, CD45, CD80, CD86, CD117, CD141, CD178, B7-H2,HLA-G, and HLA-DR, DP, DQ cell surface markers, as detected by flowcytometry. Particularly preferred are cells that produce at least two oftissue factor, vimentin, and alpha-smooth muscle actin. Also preferredare those cells producing all three of the proteins tissue factor,vimentin, and alpha-smooth muscle actin.

In other embodiments, the UTC may be characterized by gene expression,which relative to a human cell that is a fibroblast, a mesenchymal stemcell, or an iliac crest bone marrow cell, is increased for a geneencoding at least one of: interleukin 8; reticulon 1; chemokine (C-X-Cmotif) ligand 1 (melonoma growth stimulating activity, alpha); chemokine(C-X-C motif) ligand 6 (granulocyte chemotactic protein 2); chemokine(C-X-C motif) ligand 3; and tumor necrosis factor, alpha-induced protein3.

In yet other embodiments, the UTC may be characterized by geneexpression, which relative to a human cell that is a fibroblast, amesenchymal stem cell, or an iliac crest bone marrow cell, is reducedfor a gene encoding at least one of: short stature homeobox 2; heatshock 27 kDa protein 2; chemokine (C-X-C motif) ligand 12 (stromalcell-derived factor 1); elastin (supravalvular aortic stenosis,Williams-Beuren syndrome); Homo sapiens mRNA; cDNA DKFZp586M2022 (fromclone DKFZp586M2022); mesenchyme homeo box 2 (growth arrest-specifichomeo box); sine oculis homeobox homolog 1 (Drosophila); crystallin,alpha B; disheveled associated activator of morphogenesis 2;DKFZP586B2420 protein; similar to neuralin 1; tetranectin (plasminogenbinding protein); src homology three (SH3) and cysteine rich domain;cholesterol 25-hydroxylase; runt-related transcription factor 3;interleukin 11 receptor, alpha; procollagen C-endopeptidase enhancer;frizzled homolog 7 (Drosophila); hypothetical gene BC008967; collagen,type VIII, alpha 1; tenascin C (hexabrachion); iroquois homeobox protein5; hephaestin; integrin, beta 8; synaptic vesicle glycoprotein 2;neuroblastoma, suppression of tumorigenicity 1; insulin-like growthfactor binding protein 2, 36 kDa; Homo sapiens cDNA F1112280 fis, cloneMAMMA1001744; cytokine receptor-like factor 1; potassiumintermediate/small conductance calcium-activated channel, subfamily N,member 4; integrin, beta 7; transcriptional co-activator withPDZ-binding motif (TAZ); sine oculis homeobox homolog 2 (Drosophila);KIAA1034 protein; vesicle-associated membrane protein 5 (myobrevin);EGF-containing fibulin-like extracellular matrix protein 1; early growthresponse 3; distal-less homeo box 5; hypothetical protein F1120373;aldo-keto reductase family 1, member C3 (3-alpha hydroxysteroiddehydrogenase, type II); biglycan; transcriptional co-activator withPDZ-binding motif (TAZ); fibronectin 1; proenkephalin; integrin,beta-like 1 (with EGF-like repeat domains); Homo sapiens mRNA fulllength insert cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367 protein;natriuretic peptide receptor C/guanylate cyclase C (atrionatriureticpeptide receptor C); hypothetical protein FLJ14054; Homo sapiens mRNA;cDNA DKFZp564B222 (from clone DKFZp564B222); BCL2/adenovirus E1B 19 kDainteracting protein 3-like; AE binding protein 1; and cytochrome coxidase subunit VIIa polypeptide 1 (muscle).

In other embodiments, the UTC may be characterized when cultured bysecretion of at least one of MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF,HB-EGF, BDNF, TPO, MIP1b, 1309, MDC RANTES, and TIMP1. In addition, theUTC may be characterized when cultured by lack of secretion of at leastone of TGF-beta2, ANG2, PDGFbb, MIP1A, and VEGF.

In some embodiments, the UTC are derived from umbilical cord tissuesubstantially free of blood, are capable of self-renewal and expansionin culture, require L-valine for growth, can grow in at least about 5%oxygen, and comprise at least one of the following characteristics:potential for at least about 40 doublings in culture; attachment andexpansion on a coated or uncoated tissue culture vessel that comprises acoating of gelatin, laminin, collagen, polyornithine, vitronectin, orfibronectin; production of vimentin and alpha-smooth muscle actin;production of CD10, CD13, CD44, CD73, and CD90; and, expression of agene, which relative to a human cell that is a fibroblast, a mesenchymalstem cell, or an iliac crest bone marrow cell, is increased for a geneencoding interleukin 8 and reticulon 1. In some embodiments, such UTCdoes not produce CD45 and CD117.

In preferred embodiments, the cell comprises two or more of theabove-listed growth, protein/surface marker production, gene expression,or substance-secretion characteristics. More preferred is a cellcomprising three, four, five, or more of the characteristics. Still morepreferred is a UTC comprising six, seven, eight, or more of thecharacteristics. More preferred is a cell comprising all of abovecharacteristics.

Among cells that are presently preferred for use with the invention inseveral of its aspects are postpartum cells having the characteristicsdescribed above and more particularly those wherein the cells havenormal karyotypes and maintain normal karyotypes with passaging, andfurther wherein the cells express each of the markers CD10, CD13, CD44,CD73, CD90, PDGFr-alpha, and HLA-A, B, C, wherein the cells produce theimmunologically-detectable proteins which correspond to the listedmarkers. Still more preferred are those cells which in addition to theforegoing do not produce proteins corresponding to any of the markersCD31, CD34, CD45, CD117, CD141, or HLA-DR,DP,DQ, as detected by flowcytometry.

In an embodiment, the UTC are isolated from human umbilical cord tissuesubstantially free of blood, are capable of self-renewal and expansionin culture, have the potential to differentiate, lack the production ofCD117 or CD45, express CD10 and CD13, and do not express hTERT ortelomerase. These UTC optionally express oxidized low densitylipoprotein receptor 1, reticulon, chemokine receptor ligand 3, and/orgranulocyte chemotactic protein; and/or do not express CD31 or CD34;and/or express, relative to a human fibroblast, mesenchymal stem cell,or iliac crest bone marrow cell, increased levels of interleukin 8 orreticulon 1; and/or express CD44, CD73, and CD90.

In another embodiment, the UTC are isolated from human umbilical cordtissue substantially free of blood, are capable of self-renewal andexpansion in culture, have the potential to differentiate, express CD10,CD13, CD90, and HLA-ABC, and do not express CD34, CD45, CD117, andHLA-DR. Optionally, these cells also do not express hTERT or telomerase.In one embodiment, the cells also express CD44, and CD43. In yet anotherembodiment, the cells also do not express CD31. These UTC optionally:(i) express oxidized low density lipoprotein receptor 1, reticulon,chemokine receptor ligand 3, and/or granulocyte chemotactic protein;and/or (ii) express, relative to a human fibroblast, mesenchymal stemcell, or iliac crest bone marrow cell, increased levels of interleukin 8or reticulon 1.

In an alternate embodiment, the UTC are isolated from human umbilicalcord tissue substantially free of blood, are capable of self-renewal andexpansion in culture, have the potential to differentiate, and have thefollowing characteristics: (1) express CD10, CD13, CD44, CD90, andHLA-ABC; (2) do not express CD31, CD34, CD45, HLA-DR and CD117, and (3)do not express hTERT or telomerase. In another embodiment, the UTC areisolated from human umbilical cord tissue substantially free of blood,are capable of self-renewal and expansion in culture, have the potentialto differentiate, and have the following characteristics: (1) expressCD10, CD13, CD44, CD90, and HLA-ABC; (2) do not express CD31, CD34,CD45, HLA-DR and CD117; (3) do not express hTERT or telomerase; (4)express oxidized low density lipoprotein receptor 1, reticulon,chemokine receptor ligand 3, and/or granulocyte chemotactic protein; and(5) express, relative to a human fibroblast, mesenchymal stem cell, oriliac crest bone marrow cell, increased levels of interleukin 8 orreticulon 1.

In one embodiment, the hUTC are provided as a population of cells, whichmay be homogenous. In some embodiments, the cell population may beheterogeneous. A heterogeneous cell population of the invention maycomprise at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or 95% UTC of the invention. The heterogeneous cell populations of theinvention may further comprise stem cells or other progenitor cells,such as myoblasts or other muscle progenitor cells, hemangioblasts, orblood vessel precursor cells; or it may further comprise fullydifferentiated skeletal muscle cells, smooth muscle cells, pericytes, orblood vessel endothelial cells. In some embodiments, the population issubstantially

Additionally, as used in the following examples and elsewhere in thespecification, the hUTC which may be identified using the screeningmethods may be isolated and characterized according to the disclosure ofU.S. Pat. Nos. 7,510,873; 7,524,489; and U.S. Pub. App. No.2005/0058631, which are incorporated by reference in their entireties asthey relate to the description, isolation and characterization of hUTC.Furthermore, enrichment procedures using magnetic beads are described inU.S. Pat. No. 7,863,012 and U.S. Published Application No. 2011/0147180,which are being incorporated in their entireties as they relate toenrichment procedures and magnetic beads.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the present invention andpractice the claimed methods. The following working examples therefore,specifically point out the preferred embodiments of the presentinvention, and are not to be construed as limiting in any way theremainder of the disclosure.

EXAMPLES Example 1 Isolation, Maintenance, and Expansion of hUTC

Human umbilical cord tissue-derived-cells were isolated from four donorsand propagated in growth medium supplemented with 15% fetal bovine serumas described above in U.S. Pat. Nos. 7,510,873; 7,524,489; and U.S. Pub.App. No. 2005/0058631 and in the Examples below. Early passage cultureswere cryopreserved to generate development and working cell banks,termed DCB and WCB, respectively. Live cultures of hUTC were maintainedby using one of the following two methods: 1) as adherent cultures intissue culture flasks; and 2) as suspension cultures in spinner flasksand stirred tank bioreactors. Cells were first seeded onto microcarriersfor the latter method.

Three sets of samples were subjected to microarray analysis as follows:(1) Study 1 (Donor microarray study); (2) Study 2 (Temperature excursionstudy); and (3) Study 3 (Biomarker study).

Study 1 (Donor Microarray Study)

In this set of samples, cells were cultured in spinner flasks, startingfrom an early passage cell line population doubling PDL 5 and endingwith PDL44. A typical culture was initiated by seeding fresh growthmedium with an inoculum of about 5×10⁵ cells/ml and left undisturbed for4 days, at which time a peak viable cell density (VCD of about 3−4×10⁶cells/ml) was achieved. Aliquots of cultures were harvestedperiodically, for a total of thirty-two (32) samples. Table 1-1 belowlists the samples in the study.

TABLE 1-1 List of samples included in Study 1 (Donor microarray study)Label Name Donor Number R52550 Donor 1; PDL = 11.30 Umb 041505 R52551Donor 1; PDL = 20.26 Umb 041505 R52552 Donor 1; PDL = 30.06 Umb 041505R52553 Donor 1; PDL = 33.58 Umb 041505 R52554 Donor 1; PDL = 36.78 Umb041505 R52555 Donor 1; PDL = 40.03 Umb 041505 R52556 Donor 1; PDL =43.00 Umb 041505 R52557 Donor 1; PDL = 44.98 Umb 041505 R52559 Donor 2;PDL = 11.30 Umb 072304A R52560 Donor 2; PDL = 20.92 Umb 072304A R52561Donor 2; PDL = 30.29 Umb 072304A R52562 Donor 2; PDL = 31.13 Umb 072304AR52563 Donor 2; PDL = 32.66 Umb 072304A R52564 Donor 2; PDL = 33.01 Umb072304A R52565 Donor 2; PDL = 33.81 Umb 072304A R52558 Donor 2; PDL =5.00 Umb 072304A R52567 Donor 3; PDL = 10.79 Umb 083105 R52568 Donor 3;PDL = 20.12 Umb 083105 R52569 Donor 3; PDL = 30.03 Umb 083105 R52570Donor 3; PDL = 30.45 Umb 083105 R52571 Donor 3; PDL = 31.16 Umb 083105R52572 Donor 3; PDL = 31.47 Umb 083105 R52573 Donor 3; PDL = 31.69 Umb083105

Harvesting of cells was conducted at any one of the four days of growthas shown in FIG. 1 (right hand side). From the list of 32 samples, 24were selected for microarray analysis. FIG. 1 shows the populationdoubling level (PDL) and the region of the growth curve when aparticular sample was harvested.

Study 2 (Temperature Excursion Study)

In this set of samples, cells were held at temperatures ranging from 25°C. to 42° C. for up to 18 hours before harvesting. The samples used inthis study are shown in Table 1-2 below.

TABLE 1-2 List of samples included in Study 2 (Temperature excursionstudy) Label Name Donor R52550 Donor 1; PDL = 11.30 Umb 041505 R52551Donor 1; PDL = 20.26 Umb 041505 R52552 Donor 1; PDL = 30.06 Umb 041505R52553 Donor 1; PDL = 33.58 Umb 041505 R52554 Donor 1; PDL = 36.78 Umb041505 R52555 Donor 1; PDL = 40.03 Umb 041505 R52556 Donor 1; PDL =43.00 Umb 041505 R52557 Donor 1; PDL = 44.98 Umb 01505 R52559 Donor 2;PDL = 11.30 Umb 072304A R52560 Donor 2; PDL = 20.92 Umb 072304A R52561Donor 2; PDL = 30.29 Umb 072304A R52562 Donor 2; PDL = 31.13 Umb 072304AR52563 Donor 2; PDL = 32.66 Umb 072304A R52564 Donor 2; PDL = 33.01 Umb072304A R52565 Donor 2; PDL = 33.81 Umb 072304A R52558 Donor 2; PDL =5.00 Umb 072304A R52567 Donor 3; PDL = 10.79 Umb 083105 R52568 Donor 3;PDL = 20.12 Umb 083105 R52569 Donor 3; PDL = 30.03 Umb 083105 R52570Donor 3; PDL = 30.45 Umb 083105 R52571 Donor 3; PDL = 31.16 Umb 083105R52572 Donor 3; PDL = 31.47 Umb 083105 R52573 Donor 3; PDL = 31.69 Umb083105 R52566 Donor 3; PDL = 5.00 Umb 0105 R52575 Donor 4; PDL = 10.71Umb 090304A R52576 Donor 4; PDL = 14.66 Umb 090304A R52577 Donor 4; PDL= 19.55 Umb 090304A R52578 Donor 4; PDL = 25.06 Umb 090304A R52579 Donor4; PDL = 27.25 Umb 090304A R52580 Donor 4; PDL = 23.60 Umb 090304AR52581 Donor 4; PDL = 30.15 Umb 090304A R52574 Donor 4; PDL = 5.00 Umb090304A

Study 3 (Biomarker Study)

For this study, a set of eight (8) samples were used. The cells wereexpanded in a stirred tank bioreactor and harvested at PDL30. Thesamples used in this study are shown in Table 1-3 below.

TABLE 1-3 List of samples included in Study 3 (Biomarker study) Exp. IDExp. Name E55465 Donor 1_DCB_Early Passage PDL 12_Set1 E55466 Donor1_DCB_Early Passage PDL 12_Set2 E55467 Donor 1_DCB_Late Passage PDL40_Set1 E55468 Donor 1_DCB_Late Passage PDL 40_Set2 E55469 Donor1_WCB_Early Passage PDL 16_Set1 E55470 Donor 1_WCB_Early Passage PDL16_Set2 E55471 Donor 1_WCB_Late Passage PDL 36_Set1 E55472 Donor1_WCB_Late Passage PDL 36_Set2Data Analysis

An Affymetrix GeneChip® HT HG-U133+ PM array was used for microarraydata generation. The data was analyzed in two stages. In the first step,the microarray data generated from the temperature excursion study(Study 2, Table 1-2) to compare the expression profile of hUTC with thatof human PBMC. The expression level of various genes in PBMC, wasobtained from public sources, including, the National Center forBiotechnology (NCBI) database, administered by the National Institutesof Health. In the second step, the microarray data generated from Study1 and archived data from eight (8) samples generated in Study 3 (seeTables 1-1 and 1-3) were used. The expression profile of hUTC wascompared with that of (1) human PBMC and (2) rat PBMC. The expressionprofile of human PBMC was obtained from healthy human subjects (NCBIdatabase study GSE14642), while the expression profile of rat PBMC wasobtained from NCBI database studies GSE11083 and GSE19537. Expressionprofiles of subtypes of human blood cells (B cell, NK cell, dendriticcell, lymphoid T cell, myeloid monocyte and neutrophils) were alsoobtained from NCBI databases (study GSE22886 and study GSE3982).Additionally, mining the Gene Ontology and Entrez databases identified3976 human plasma membrane protein genes (GO:0005886), 355 cell surfaceprotein genes (GO:0009986) and 349 CD antigen genes. A subset of 2614human and rat plasma membrane protein genes and 288 cell surface proteingenes were matched to genes on the human and rat microarrays and 315 CDantigen genes and were matched to human microarrays were included forthis study.

Based on the dynamics of the expression signal, i.e., log 2 (intensity)of greater or equal to ten as threshold for high expression signals forall the datasets, a list of 314 probe sets from over 200 genes that arehighly expressed in both types of samples were identified. In bothcases, genes whose expression in hUTC was dramatically different fromthat of human PBMC were selected and rank ordered. Table 1-4 shows thelist of genes, generated in Study 2, whose expression levels weresignificantly different between hUTC and that of human PBMC. Theseinclude cell surface and plasma membrane proteins as well asintracellular proteins.

In Table 1-4, genes that were identified in all three sets of samplesare underlined. Genes that were examined in more detail are bolded (theyinclude DKK3, LAMB1, ANPEP (CD13), LAMP1 (CD107a), PTPRC(CD45), MME(CD10) and NRP1).

TABLE 1-4 List of genes whose expression levels were significantlydifferent between hUTC and that of human PBMC in Study 2 samples EntrezGene Symbol Gene Gene Title KRT19 3880 Keratin 19 DKK3 27122 Dickkopfhomolog 3 (Xenopus laevis) GJA1 2697 Gap junction protein, alpha 1, 43kDa LOX 4015 Lysyl oxidase FBN1 2200 Fibrillin 1 COL3A1 1281 Collagen,type III, alpha 1 TPM2 7169 Tropomyosin 2 (beta) COL1A1 1277 Collagen,type I, alpha 1 CYR61 3491 Cysteine-rich, angiogenic inducer, 61 DKK122943 Dickkopf homolog 1 (Xenopus laevis) CTGF 1490 Connective tissuegrowth factor DCBLD2 131566 Discoidin, CUB and LCCL domain containingITGB5 3693 Integrin, beta 5 COL5A1 1289 Collagen, type V, alpha 1 THY17070 Thy-1 cell surface antigen FTL 2512 Ferritin, light polypeptideNNMT 4837 Nicotinamide N-methyltransferase TMEM47 83604 Transmembraneprotein 47 CDH11 1009 Cadherin 11, type 2, OB-cadherin (osteoblast)SPTBN1 6711 Spectrin, beta, non-erythrocytic 1 RPS19 6223 Ribosomalprotein S19 DCN 1634 Decorin RPS20 6224 Ribosomal protein S20 LAMB1 3912Laminin, beta 1 COL5A2 1290 Collagen, type V, alpha 2 SERPINE2 5270Terpin peptidase inhibitor, clade E (nexin) TPBG 7162 Trophoblastglycoprotein CLMP 79827 CXADR-like membrane protein MAP1B 4131Microtubule-associated protein 1B SLIT2 9353 Slit homolog 2 (Drosophila)FRMD6 122786 FERM domain containing 6 CSPG4 1464 Chondroitin sulfateproteoglycan 4 PLOD2 5352 Procollagen-lysine, 2-oxoglutarate5-dioxygenase 2 CDH2 1000 Cadherin 2, type 1, N-cadherin (neuronal)COL1A2 1278 Collagen, type I, alpha 2 FAT1 2195 FAT tumor suppressorhomolog 1 (Drosophila) GNG12 55970 Guanine nucleotide binding protein (Gprotein), gamma 12 FGG 2266 Fibrinogen gamma chain SULF1 23213 Sulfatase1 ANTXR1 84168 Anthrax toxin receptor 1 PAPPA 5069 PAPPA antisense RNA(non-protein coding) MFAP5 8076 Microfibrillar associated protein 5SSTR1 6751 Somatostatin receptor 1 CAP2 10486 CAP, adenylatecyclase-associated protein, EDIL3 10085 EGF-like repeats and discoidinI-like domains TEK 7010 TEK tyrosine kinase, endothelial YAP1 10413Yes-associated protein 1 PTRF 284119 Polymerase I and transcript releasefactor LUM 4060 lumican WWTR1 25937 WW domain containing transcriptionregulator NR2F2 7026 nuclear receptor subfamily 2, group F, member ANPEP290 alanyl (membrane) aminopeptidase (CD13) LAMP1 3916lysosomal-associated membrane protein 1 (CD107a) PTPRC (CD45) 5788protein tyrosine phosphatase, receptor type, C MME (CD10) 4311 matrixmetallo proteasr NRP1 8829 neuropilin 1

Table 1-5 shows the list of genes (generated in Study 1 and Study 2),whose expression levels were significantly different between hUTC andthat of human PBMC. In Table 1-5, underlining indicates genes that wereidentified in all three sets of samples, i.e., Study 1, 2, and 3 (theyinclude LAMB1, DKK3 and CAP2). Genes that were examined in more detailare bolded (they include ANPEP (CD13), LAMP1 (CD107a), PTPRC(CD45), MME(CD10) and NRP1).

TABLE 1-5 List of genes whose expression levels were significantlydifferent between hUTC and that of human PBMC in samples generated forStudy 2 and 3 Entrees Gene Symbol Gene Gene Title LAMB1 3912 laminin,beta 1 LUM 4060 Lumican WWTR1 25937 WW domain containing transcriptionregulator DKK3 27122 dickkopf homolog 3 (Xenopus laevis) NR2F2 7026nuclear receptor subfamily 2, group F, member CAP2 10486 CAP, adenylatecyclase-associated protein, 2 ANPEP 290 alanyl (membrane) aminopeptidase(CD13) LAMP1 3916 lysosomal-associated membrane protein 1 (CD107a) PTPRC(CD45) 5788 protein tyrosine phosphatase, receptor type, C MME (CD10)4311 matrix metallo protease NRP1 8829 neuropilin 1

As shown in Table 1-5, eleven genes were identified by analysis ofsample sets comprising of Study 2 and 3, including three genes that wereidentified in all three sets of samples. Of these eleven genes, sevengenes (NRP1, DKK3, LAMP1, LAMB1, MME (CD10), PTPRC(CD45) and ANPEP(CD13)) were examined in more detail.

Of these seven genes, NRP1, DKK3, LAMP1, LAMB1 and MME (CD10) are cellsurface markers while the expressed proteins, PTPRC(CD45) and ANPEP(CD13) are localized intracellularly. The expression levels of threegenes, namely, PTPRC(CD45) (gene ID #207238), MME (CD10) (gene ID#203434) and ANPEP (CD13) (gene ID #202888) in hUTC and in various typesof human blood cells that are PBMC were compared. This comparison ofexpression of cell surface protein genes, PTPRC(CD45), MME (CD10), andANPEP (CD13) in hUTC and in various types of blood cells that comprisehuman PBMC is shown in FIGS. 2A, B, and C, respectively.

Example 2 RT-PCR Confirmation of Identified Markers

The transcription levels of selected genes in hUTC and human PBMCidentified in Example 1 were then confirmed by RT-PCR. The results ofthe RT-PCR of select genes whose expression in hUTC was compared to thatof human PBMC are shown in FIG. 3.

To obtain these results, total RNA from each hUTC preparation wasisolated from which cDNA was then prepared. Fluorescent probes specificfor each gene were then used to perform the RT-PCR reaction using thecDNA as the template.

FIG. 3 shows the results of RT-PCR for MME (CD10), ANPEP (CD13),PTPRC(CD45), DKK3, LAMB1, NRP1, GAPPD, and HPRT1 in hUTC and PBMC. Withthe exception of the housekeeping genes, GAPDH and HPRT, all the genestested show a greater abundance in hUTC as compared to human PBMC. Onlythe expression of PTPRC(CD45), which was used as a negative control andwhich is known to be expressed highly in human PBMC, was less abundantin hUTC. With reference to FIG. 3, a higher CT value is indicative of alower amount of transcript.

Since PBMC is comprised of various types of blood cells, the geneexpression profiles of select genes expressed in each of these celltypes to that of hUTC were compared. As shown in FIG. 2B, while therelative expression of MME (CD10) in PBMC is low when compared to thatof hUTC, its relative expression in neutrophil is high. Similarly, whilethe expression of ANPEP (CD13) in hUTC is comparable to that of humanPBMC (see FIG. 2C), its relative expression in T-cells and macrophagesis higher than that of hUTC.

Example 3 Detection of hUTC in PBMC by Flow Cytometry

From the list of genes that show differential expression in hUTC andhuman PBMC (see Tables 1-4 and 1-5), a subset of five genes that expressthe gene product on cell surface and/or plasma membrane were selected.Additionally, two genes that express the corresponding protein productsintracellularly were selected. These markers included NRP1, DKK3, LAMB,LAMP1, MME (CD10), PTPRC(CD45), and ANPEP (CD13).

For the flow cytometry assay, the cells were harvested in theexponential phase and subsequently, fixed and permeablized using a kitpurchased from BD Bioscience. An aliquot of this preparation was thenincubated with antibodies against selected markers identified to bepresent on hUTC surface, namely, CD10, CD13, CD45, NRP1, and LAMP1.After removing excess antibody, the cells were incubated with afluorescently labeled secondary antibody. Cells were then analyzed by aflow cytometer.

The results for the flow cytometry assay for the detection of cellsurface, plasma membrane, and intracellular markers are shown in FIGS.4A to 4C. FIG. 4A shows the cell surface markers that were tested usinghUTC with the top panel being the control. FIG. 4B shows the cellsurface markers that were tested using PBMC with the top panel being thecontrol. FIG. 4C shows the results for a flow cytometry assay for thedetection of the internal markers DKK3 and LAMP1 in PBMC and hUTC.

With reference to FIG. 4A, 90% of the hUTC population was observed to beCD13 positive (CD13⁺), 75% of the hUTC population was observed to beCD10 positive (CD10⁺), and 17% of the hUTC population was observed to beNRP1 positive (NRP1⁺). None of the hUTC population was observed to bepositive for CD45 or LAMP1.

With reference to FIG. 4B, 6% of the PBMC population was observed to beCD13 positive (CD13⁺), 2% of the PBMC population was observed to be CD10positive (CD10⁺), 4% of the PBMC population was observed to be NRP1positive (NRP1⁺), and 72% of the PBMC population was observed to be CD45positive (CD45⁺). None of the PBMC population was observed to bepositive for LAMP1.

With reference to FIG. 4C, flow cytometry data for the intracellularmarkers DKK3 and LAMP1 is shown. 52% of the hUTC population was observedto be DKK3 positive. 23% of the PBMC population was observed to be DKK3positive. FIG. 4C indicates that LAMP1 is a good internal marker forhUTC.

With the exception of NRP1, the cell surface protein gene markers couldbe used to detect intact, live hUTC in mixed samples containing PBMCs inserum (see FIGS. 4A and 4B). Even though NRP1 is transcribed at higherlevels in hUTC as compared to that of human PBMC (FIG. 3), NRP1 couldnot be detected in hUTC by flow cytometry (see FIG. 4A). With respect toCD45, as expected, high levels of CD45 were being expressed on thesurface of live human PBMC only (see FIG. 4B). Additionally, twointracellular markers LAMP1 and DKK3 (from the list of differentiallyexpressed genes, Table 1-4) whose expression is higher in hUTC ascompared to human PBMC) were also examined (see FIG. 4C). These markerscan be used as additional confirmation for hUTC identity.

The differences between hUTC and human PBMC as assayed by flow cytometryare shown in Table 3-1 below.

TABLE 3-1 Difference between hUTC and PBMC with respect to percentpositive cells as assayed by flow cytometry. Population % CD13 % CD10 %NRP1 % CD45 % LAMP1 % DKK3 % LAMB hUTC 90 70-90 17 0 92 52 ND PBMC 6 2 445 32 23 ND

Table 3-2 below summarizes the differences between hUTC and PBMC asassayed by RT-PCR in Example 2 above and by flow cytometry in theinstant Example. In Table 3-2, Nd means Not determined.

TABLE 3-2 Summary of RT-PCR and flow cytometry results Microarray FlowAnalysis Flow Analysis Analysis RT-PCR surface intracellular hUTC PBMChUTC PBMC hUTC PBMC hUTC PBMC MME (CD10) + − + − + − Nd Nd ANPEP(CD13) + + + + + − Nd Nd PTPRC (CD45) − + − + − + Nd Nd LAMP1 + + + + −− + − NRP1 + − + − − − Nd Nd DKK3 + − + − Nd Nd ++ + LAMB1 + + + − Nd NdNd Nd

Example 4 Detection of hUTC in a Mixture of hUTC and Human PBMC

To demonstrate detection of hUTC in blood, various concentrations ofhUTC were mixed with 1 million human PBMC. The mixture was then analyzedby flow cytometry using CD45 (positive marker for PBMC), CD10 and CD13(positive markers for hUTC). In particular, the detection of hUTC in amixture comprising of hUTC (ranging from 1,500 cells/ml to 110,000cells/ml) and human PBMC (1 million cells/ml) is shown in FIGS. 5A and5B.

The two types of cells were mixed in 1 ml of human serum at roomtemperature. Immediately thereafter, aliquots of the mixture wereanalyzed by flow cytometry using CD10 as a marker for hUTC and CD45 as amarker for PBMC. In FIG. 5A, the concentration of hUTC ranged from 1,500to 1,700 cells/ml and that of human PBMC was 1 million cells/ml. In FIG.5B, the concentration of hUTC ranged from 1,700 to 110,000 cells/ml andthat of human PBMC was 1 million cells/ml.

As can be seen from FIG. 5A, if the sample contains 1,700 or morehUTC/ml in the presence of 1 million human PBMC, then the accuracy ofdetermination using flow cytometry is high (R²=0.94). If the samplecontains 1,500 to 1,700 hUTC/ml in the presence of 1 million human PBMC(FIG. 5B), then the accuracy of determination using flow cytometry islower (R²=0.51).

Example 5 Isolation of hUTC

Umbilical Cell Isolation.

Umbilical cords were obtained from National Disease Research Interchange(NDRI, Philadelphia, Pa.). The tissues were obtained following normaldeliveries. The cell isolation protocols were performed aseptically in alaminar flow hood. To remove blood and debris, the cord was washed inphosphate buffered saline (PBS; Invitrogen, Carlsbad, Calif.) in thepresence of penicillin at 100 U/ml, streptomycin at 100 mg/ml andamphotericin B at 0.25 μg/ml (Invitrogen Carlsbad, Calif.). The tissueswere then mechanically dissociated in 150 cm² tissue culture plates inthe presence of 50 ml of medium (DMEM-low glucose or DMEM-high glucose;Invitrogen) until the tissue was minced into a fine pulp. The choppedtissues were transferred to 50 ml conical tubes (approximately 5 g oftissue per tube).

The tissue was then digested in either DMEM-low glucose medium orDMEM-high glucose medium, each containing penicillin at 100 U/ml,streptomycin at 100 mg/ml, amphotericin B at 0.25 μg/ml and thedigestion enzymes. In some experiments an enzyme mixture of collagenaseand dispase was used (“C:D”) (collagenase (Sigma, St Louis, Mo.), 500U/ml; and dispase (Invitrogen), 50 U/ml, in DMEM-Low glucose medium). Inother experiments a mixture of collagenase, dispase and hyaluronidase(“C:D:H”) was used (C:D:H=collagenase, 500 U/ml; dispase, 50 U/ml; andhyaluronidase (Sigma), 5 U/ml, in DMEM-Low glucose). The conical tubescontaining the tissue, medium and digestion enzymes were incubated at37° C. in an orbital shaker (Environ, Brooklyn, N.Y.) at 225 rpm for 2hours.

After digestion, the tissues were centrifuged at 150×g for 5 minutes,the supernatant was aspirated. The pellet was resuspended in 20 ml ofgrowth medium (DMEM:Low glucose (Invitrogen), 15% (v/v) fetal bovineserum (FBS; defined fetal bovine serum; Lot #AND18475; Hyclone, Logan,Utah), 0.001% (v/v) 2-mercaptoethanol (Sigma), penicillin at 100 U/ml,streptomycin at 100 μg/ml, and amphotericin B at 0.25 μg/ml (each fromInvitrogen, Carlsbad, Calif.)). The cell suspension was filtered througha 70 μm nylon BD FALCON Cell Strainer (BD Biosciences, San Jose,Calif.). An additional 5 ml rinse comprising growth medium was passedthrough the strainer. The cell suspension was then passed through a40-μm nylon cell strainer (BD Biosciences, San Jose, Calif.) and chasedwith a rinse of an additional 5 ml of growth medium.

The filtrate was resuspended in growth medium (total volume 50 ml) andcentrifuged at 150×g for 5 minutes. The supernatant was aspirated andthe cells were resuspended in 50 ml of fresh growth medium. This processwas repeated twice more.

After the final centrifugation, supernatant was aspirated and the cellpellet was resuspended in 5 ml of fresh growth medium. The number ofviable cells was determined using trypan blue staining Cells were thencultured under standard conditions.

The cells isolated from umbilical cord tissues were seeded at 5,000cells/cm² onto gelatin-coated T-75 flasks (Corning Inc., Corning, N.Y.)in growth medium. After two days, spent medium and unadhered cells wereaspirated from the flasks. Adherent cells were washed with PBS threetimes to remove debris and blood-derived cells. Cells were thenreplenished with growth medium and allowed to grow to confluence (about10 days from passage 0 to passage 1). On subsequent passages (frompassage 1 to 2 etc.), cells reached sub-confluence (75-85% confluence)in 4-5 days. For these subsequent passages, cells were seeded at 5,000cells/cm². Cells were grown in a humidified incubator with 5% carbondioxide at 37° C.

In some experiments, cells were isolated from postpartum tissues inDMEM-low glucose medium after digestion with LIBERASE (2.5 mg/ml,Blendzyme 3; Roche Applied Sciences, Indianapolis, Ind.) andhyaluronidase (5 U/ml, Sigma). Digestion of the tissue and isolation ofthe cells was as described for other protease digestions above, however,the LIBERASE/hyaluronidase mixture was used instead of the C:D or C:D:Henzyme mixture. Tissue digestion with LIBERASE resulted in the isolationof cell populations from postpartum tissues that expanded readily.

Procedures were compared for isolating cells from the umbilical cordusing differing enzyme combinations. Enzymes compared for digestionincluded: i) collagenase; ii) dispase; iii) hyaluronidase; iv)collagenase:dispase mixture (C:D); v) collagenase: hyaluronidase mixture(C:H); vi) dispase: hyaluronidase mixture (D:H); and vii)collagenase:dispase:hyaluronidase mixture (C:D:H). Differences in cellisolation utilizing these different enzyme digestion conditions wereobserved (see Table 5-1).

Other attempts were made to isolate pools of cells from umbilical cordby different approaches. In one instance, umbilical cord was sliced andwashed with growth medium to dislodge the blood clots and gelatinousmaterial. The mixture of blood, gelatinous material and growth mediumwas collected and centrifuged at 150×g. The pellet was resuspended andseeded onto gelatin coated flasks in growth medium. From theseexperiments, a cell population was isolated that readily expanded.

Cells have also been isolated from cord blood samples obtained fromNDRI. The isolation protocol used was that of International PatentApplication PCT/US2002/029971 by Ho et al. Samples (50 ml and 10.5 ml,respectively) of umbilical cord blood (NDRI, Philadelphia Pa.) weremixed with lysis buffer (filter-sterilized 155 mM ammonium chloride, 10millimolar potassium bicarbonate, 0.1 mM EDTA buffered to pH 7.2 (allcomponents from Sigma, St. Louis, Mo.)). Cells were lysed at a ratio of1:20 cord blood to lysis buffer. The resulting cell suspension wasvortexed for 5 seconds, and incubated for 2 minutes at ambienttemperature. The lysate was centrifuged (10 minutes at 200×g). The cellpellet was resuspended in Complete Minimal Essential Medium (Gibco,Carlsbad Calif.) containing 10% fetal bovine serum (Hyclone, LoganUtah), 4 mM glutamine (Mediatech Herndon, Va.), penicillin at 100 U/mland streptomycin at 100 μg/ml (Gibco, Carlsbad, Calif.). The resuspendedcells were centrifuged (10 minutes at 200×g), the supernatant wasaspirated, and the cell pellet was washed in complete medium. Cells wereseeded directly into either T75 flasks (Corning, N.Y.), T75laminin-coated flasks, or T175 fibronectin-coated flasks (both BectonDickinson, Bedford, Mass.).

To determine whether cell populations could be isolated under differentconditions and expanded under a variety of conditions immediately afterisolation, cells were digested in growth medium with or without 0.001%(v/v) 2-mercaptoethanol (Sigma, St. Louis, Mo.), using the enzymecombination of C:D:H, according to the procedures provided above. Allcells were grown in the presence of penicillin at 100 U/ml andstreptomycin at 100 μg/ml. Under all tested conditions, cells attachedand expanded well between passage 0 and 1 (Table 4-2). Cells inconditions 5-8 and 13-16 were demonstrated to proliferate well up to 4passages after seeding, at which point they were cryopreserved.

The combination of C:D:H, provided the best cell yield followingisolation, and generated cells that expanded for many more generationsin culture than the other conditions (Table 5-1). An expandable cellpopulation was not attained using collagenase or hyaluronidase alone. Noattempt was made to determine if this result is specific to thecollagenase that was tested.

TABLE 5-1 Isolation of cells from umbilical cord tissue using varyingenzyme combinations Enzyme Digest Cells Isolated Cell ExpansionCollagenase X X Dispase + (>10 h) + Hyaluronidase X XCollagenase:Dispase ++ (<3 h) ++ Collagenase:Hyaluronidase ++ (<3 h) +Dispase:Hyaluronidase + (>10 h) + Collagenase:Dispase:Hyaluronidase +++(<3 h) +++ Key: + = good, ++ = very good, +++ = excellent, X = nosuccess

Cells attached and expanded well between passage 0 and 1 under allconditions tested for enzyme digestion and growth (Table 5-2). Cells inexperimental conditions 5-8 and 13-16 proliferated well up to fourpassages after seeding, at which point they were cryopreserved. Allcells were cryopreserved for further analysis.

TABLE 5-2 Isolation and culture expansion of postpartum cells undervarying conditions Condition Medium 15% FBS BME Gelatin 20% O₂ GrowthFactors 1 DMEM-Lg Y Y Y Y N 2 DMEM-Lg Y Y Y N (5%) N 3 DMEM-Lg Y Y N Y N4 DMEM-Lg Y Y N N (5%) N 5 DMEM-Lg N (2%) Y N (Laminin) Y EGF/FGF (20ng/ml) 6 DMEM-Lg N (2%) Y N (Laminin) N (5%) EGF/FGF (20 ng/ml) 7DMEM-Lg N (2%) Y N (Fibronectin) Y PDGF/VEGF 8 DMEM-Lg N (2%) Y N(Fibronectin) N (5%) PDGF/VEGF 9 DMEM-Lg Y N Y Y N 10 DMEM-Lg Y N Y N(5%) N 11 DMEM-Lg Y N N Y N 12 DMEM-Lg Y N N N (5%) N 13 DMEM-Lg N (2%)N N (Laminin) Y EGF/FGF (20 ng/ml) 14 DMEM-Lg N (2%) N N (Laminin) N(5%) EGF/FGF (20 ng/ml) 15 DMEM-Lg N (2%) N N (Fibronectin) Y PDGF/VEGF16 DMEM-Lg N (2%) N N (Fibronectin) N (5%) PDGF/VEGF

Nucleated cells attached and grew rapidly. These cells were analyzed byflow cytometry and were similar to cells obtained by enzyme digestion.

The preparations contained red blood cells and platelets. No nucleatedcells attached and divided during the first 3 weeks. The medium waschanged 3 weeks after seeding and no cells were observed to attach andgrow.

Populations of cells could be isolated from umbilical tissue efficientlyusing the enzyme combination collagenase (a metalloprotease), dispase(neutral protease) and hyaluronidase (mucolytic enzyme which breaks downhyaluronic acid). LIBERASE, which is a blend of collagenase and aneutral protease, may also be used. Blendzyme 3, which is collagenase (4Wunsch U/g) and thermolysin (1714 casein U/g), was also used togetherwith hyaluronidase to isolate cells. These cells expanded readily overmany passages when cultured in growth expansion medium on gelatin coatedplastic.

Cells were also isolated from residual blood in the cords, but not cordblood. The presence of cells in blood clots washed from the tissue,which adhere and grow under the conditions used, may be due to cellsbeing released during the dissection process.

Example 6 Karyotype Analysis of Cells

Cell lines used in cell therapy are preferably homogeneous and free fromany contaminating cell type. Human cells used in cell therapy shouldhave a normal number (46) of chromosomes with normal structure. Toidentify umbilicus-derived cell lines that are homogeneous and free fromcells of non-umbilical tissue origin, karyotypes of cell samples wereanalyzed.

UTC from postpartum tissue of a male neonate were cultured in growthmedia. Postpartum tissue from a male neonate (X,Y) was selected to allowdistinction between neonatal-derived cells and maternal derived cells(X,X). Cells were seeded at 5,000 cells per square centimeter in growthmedium in a T25 flask (Corning, Corning, N.Y.) and expanded to 80%confluence. A T25 flask containing cells was filled to the neck withgrowth media. Samples were delivered to a clinical cytogenetics lab bycourier (estimated lab to lab transport time is one hour). Chromosomeanalysis was performed by the Center for Human & Molecular Genetics atthe New Jersey Medical School, Newark, N.J. Cells were analyzed duringmetaphase when the chromosomes are best visualized. Of twenty cells inmetaphase counted, five were analyzed for normal homogeneous karyotypenumber (two). A cell sample was characterized as homogeneous if twokaryotypes were observed. A cell sample was characterized asheterogeneous if more than two karyotypes were observed. Additionalmetaphase cells were counted and analyzed when a heterogeneous karyotypenumber (four) was identified.

All cell samples sent for chromosome analysis were interpreted by thecytogenetics laboratory staff as exhibiting a normal appearance. Threeof the sixteen cell lines analyzed exhibited a heterogeneous phenotype(XX and XY) indicating the presence of cells derived from both neonataland maternal origins (Table 6-1). Each of the cell samples wascharacterized as homogeneous. (Table 6-1).

TABLE 6-1 Karyotype results of hUTC Metaphase Metaphase cells cellsNumber of ISCN Tissue Passage counted analyzed karyotypes KaryotypeUmbilical 23 20 5 2 46, XX Umbilical 6 20 5 2 46, XY Umbilical 3 20 5 246, XX

Chromosome analysis identified umbilicus-derived UTC whose karyotypesappear normal as interpreted by a clinical cytogenetic laboratory.Karyotype analysis also identified cell lines free from maternal cells,as determined by homogeneous karyotype.

Example 7 Flow Cytometric Evaluation of Cell Surface Markers

Characterization of cell surface proteins or “markers” by flow cytometrycan be used to determine a cell line's identity. The consistency ofexpression can be determined from multiple donors, and in cells exposedto different processing and culturing conditions. Postpartum cell linesisolated from the umbilicus were characterized by flow cytometry,providing a profile for the identification of these cell lines.

Cells were cultured in growth medium, in plasma-treated T75, T150, andT225 tissue culture flasks (Corning, Corning, N.Y.) until confluent. Thegrowth surfaces of the flasks were coated with gelatin by incubating 2%(w/v) gelatin (Sigma, St. Louis, Mo.) for 20 minutes at roomtemperature.

Adherent cells in flasks were washed in phosphate buffered saline (PBS);(Gibco, Carlsbad, Mo.) and detached with trypsin/EDTA (Gibco). Cellswere harvested, centrifuged, and resuspended in 3% (v/v) FBS in PBS at acell concentration of 1×10⁷/ml. In accordance with the manufacture'sspecifications, antibody to the cell surface marker of interest (seebelow) was added to 100 μl of cell suspension and the mixture wasincubated in the dark for 30 minutes at 4° C. After incubation, cellswere washed with PBS and centrifuged to remove unbound antibody. Cellswere resuspended in 500 μl PBS and analyzed by flow cytometry. Flowcytometry analysis was performed with a FACS calibur instrument (BectonDickinson, San Jose, Calif.).

The following antibodies to cell surface markers were used.

TABLE 7-1 Antibodies used in characterizing cell surface markers ofUDCs. Catalog Antibody Manufacturer Number CD10 BD Pharmingen (SanDiego, CA) 555375 CD13 BD Pharmingen 555394 CD31 BD Pharmingen 555446CD34 BD Pharmingen 555821 CD44 BD Pharmingen 555478 CD45RA BD Pharmingen555489 CD73 BD Pharmingen 550257 CD90 BD Pharmingen 555596 CD117 BDPharmingen 340529 CD141 BD Pharmingen 559781 PDGFr-alpha BD Pharmingen556002 HLA-A, B, C BD Pharmingen 555553 HLA-DR, DP, DQ BD Pharmingen555558 IgG-FITC Sigma (St. Louis, MO) F-6522 IgG-PE Sigma P-4685

Umbilicus-derived cells were analyzed at passages 8, 15, and 20.

To compare differences among donors, umbilical cord tissue-derived cellsfrom different donors were compared to each other. Umbilicus-derivedcells cultured on gelatin-coated flasks were also compared toumbilicus-derived cells cultured on uncoated flasks.

Four treatments used for isolation and preparation of cells werecompared. Cells derived from postpartum tissue by treatment with: 1)collagenase; 2) collagenase/dispase; 3) collagenase/hyaluronidase; and4) collagenase/hyaluronidase/dispase were compared.

Umbilical cord-derived cells at passage 8, 15, and 20 analyzed by flowcytometry all expressed CD10, CD13, CD44, CD73, CD90, PDGFr-alpha andHLA-A, B, C, indicated by increased fluorescence relative to the IgGcontrol. These cells were negative for CD31, CD34, CD45, CD117, CD141,and HLA-DR, DP, DQ, indicated by fluorescence values consistent with theIgG control.

Umbilical cord-derived cells isolated from separate donors analyzed byflow cytometry each showed positive for the production of CD10, CD13,CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B, C, reflected in theincreased values of fluorescence relative to the IgG control. Thesecells were negative for the production of CD31, CD34, CD45, CD117,CD141, and HLA-DR, DP, DQ with fluorescence values consistent with theIgG control.

The umbilical cord-derived cells expanded on gelatin-coated and uncoatedflasks analyzed by flow cytometry were all positive for the productionof CD10, CD13, CD44, CD73, CD 90, PDGFr-alpha, and HLA-A, B, C, withincreased values of fluorescence relative to the IgG control. Thesecells were negative for the production of CD31, CD34, CD45, CD117,CD141, and HLA-DR, DP, DQ, with fluorescence values consistent with theIgG control.

Analysis of umbilical cord-derived cells by flow cytometry hasestablished an identity of these cell lines. These umbilicalcord-derived cells are positive for CD10, CD13, CD44, CD73, CD90,PDGFr-alpha, and HLA-A,B,C; and negative for CD31, CD34, CD45, CD117,CD141 and HLA-DR, DP, DQ. This identity was consistent betweenvariations in variables including the donor, passage, culture vesselsurface coating, digestion enzymes, and placental layer. Some variationin individual fluorescence value histogram curve means and ranges wereobserved, but all positive curves under all conditions tested werenormal and expressed fluorescence values greater than the IgG control,thus confirming that the cells comprise a homogeneous population, whichhas positive expression of the markers.

Example 8 Analysis of Cells by Oligonucleotide Array

Oligonucleotide arrays were used to compare gene expression profiles ofumbilicus-derived and placenta-derived cells with fibroblasts, humanmesenchymal stem cells, and another cell line derived from human bonemarrow. This analysis provided a characterization of thepostpartum-derived cells and identified unique molecular markers forthese cells.

Postpartum Tissue-Derived Cells.

Human umbilical cords and placenta were obtained from National DiseaseResearch Interchange (NDRI, Philadelphia, Pa.) from normal full termdeliveries with patient consent. The tissues were received and cellswere isolated as described in Example 5 after digestion with a C:D:Hmixture. The cells were cultured in growth medium on gelatin-coatedplastic tissue culture flasks. The cultures were incubated at 37° C.with 5% CO₂.

Fibroblasts.

Human dermal fibroblasts were purchased from Cambrex Incorporated(Walkersville, Md.; Lot number 9F0844) and ATCC CRL-1501 (CCD39SK). Bothlines were cultured in DMEM/F12 medium (Invitrogen, Carlsbad, Calif.)with 10% (v/v) fetal bovine serum (Hyclone) and penicillin/streptomycin(Invitrogen)). The cells were grown on standard tissue-culture treatedplastic.

Human Mesenchymal Stem Cells (hMSC).

hMSCs were purchased from Cambrex Incorporated (Walkersville, Md.; Lotnumbers 2F1655, 2F1656 and 2F1657) and cultured according to themanufacturer's specifications in MSCGM Media (Cambrex). The cells weregrown on standard tissue cultured plastic at 37° C. with 5% CO₂.

Human Iliac Crest Bone Marrow Cells (ICBM).

Human iliac crest bone marrow was received from NDRI with patientconsent. The marrow was processed according to the method outlined byHo, et al. (WO03/025149). The marrow was mixed with lysis buffer (155 mMNH₄Cl, 10 mM KHCO₃, and 0.1 mM EDTA, pH 7.2) at a ratio of 1 part bonemarrow to 20 parts lysis buffer. The cell suspension was vortexed,incubated for 2 minutes at ambient temperature, and centrifuged for 10minutes at 500×g. The supernatant was discarded and the cell pellet wasresuspended in Minimal Essential Medium-alpha (Invitrogen) supplementedwith 10% (v/v) fetal bovine serum and 4 mM glutamine. The cells werecentrifuged again and the cell pellet was resuspended in fresh medium.The viable mononuclear cells were counted using trypan blue exclusion(Sigma, St. Louis, Mo.). The mononuclear cells were seeded in plastictissue culture flasks at 5×10⁴ cells/cm². The cells were incubated at37° C. with 5% CO₂ at either standard atmospheric O₂ or at 5% O₂. Cellswere cultured for 5 days without a media change. Media and non-adherentcells were removed after 5 days of culturing. The adherent cells weremaintained in culture.

Actively growing cultures of cells were removed from the flasks with acell scraper in cold phosphate buffered saline (PBS). The cells werecentrifuged for 5 minutes at 300×g. The supernatant was removed and thecells were resuspended in fresh PBS and centrifuged again. Thesupernatant was removed and the cell pellet was immediately frozen andstored at −80° C. Cellular mRNA was extracted and transcribed into cDNA.The cDNA was then transcribed into cRNA and biotin-labeled. Thebiotin-labeled cRNA was hybridized with Affymetrix GENECHIP HG-U133Aoligonucleotide arrays (Affymetrix, Santa Clara, Calif.). Thehybridizations and data collection were performed according to themanufacturer's specifications. Data analysis was performed using“Significance Analysis of Microarrays” (SAM) version 1.21 computersoftware (Tusher, V. G. et al., 2001, Proc. Natl. Acad. Sci. USA 98:5116-5121). Licenses for the analysis software are available through theOffice of Technology Licensing, Stanford University, and moreinformation is available on the World Wide Web at Professor Tibshirani'sweb site in the Dep't of Statistics, Stanford University.

Fourteen different populations of cells were analyzed in this study. Thecells, along with passage information, culture substrate, and culturemedia are listed in Table 8-1. The cells lines are listed by theiridentification code along with passage at the time of analysis, cellgrowth substrate, and growth media.

TABLE 8-1 Cells analyzed by the microarray study. Cell PopulationPassage Substrate Media Umbilical (022803) 2 Gelatin DMEM, 15% FBS,2-BME Umbilical (042103) 3 Gelatin DMEM, 15% FBS, 2-BME Umbilical(071003) 4 Gelatin DMEM, 15% FBS, 2-BME Placenta (042203) 12 GelatinDMEM, 15% FBS, 2-BME Placenta (042903) 4 Gelatin DMEM, 15% FBS, 2-BMEPlacenta (071003) 3 Gelatin DMEM, 15% FBS, 2-BME ICBM (070203) 3 PlasticMEM 10% FBS (5% O₂) ICBM (062703) 5 Plastic MEM 10% FBS (std. O₂) ICBM(062703) 5 Plastic MEM 10% FBS (5% O₂) hMSC (Lot 2F1655) 3 Plastic MSCGMhMSC (Lot 2F1656) 3 Plastic MSCGM hMSC (Lot 2F1657) 3 Plastic MSCGMhFibroblast (9F0844) 9 Plastic DMEM-F12, 10% FBS hFibroblast 4 PlasticDMEM-F12, 10% FBS (CCD39SK)

The data were evaluated by principle component analysis with SAMsoftware as described above. The analysis revealed 290 genes that wereexpressed in different relative amounts in the cells tested. Thisanalysis provided relative comparisons between the populations.

Table 8-2 shows the Euclidean distances that were calculated for thecomparison of the cell pairs. The Euclidean distances were based on thecomparison of the cells based on the 290 genes that were differentiallyexpressed among the cell types. The Euclidean distance is inverselyproportional to similarity between the expression of the 290 genes. TheEuclidean distance was calculated for the cell types using these 290genes expressed differentially between the cell types. Similaritybetween the cells is inversely proportional to the Euclidean distance.

TABLE 8-2 The Euclidean Distances for the Cell Pairs. Cell PairEuclidean Distance ICBM-hMSC 24.71 Placenta-umbilical 25.52ICBM-Fibroblast 36.44 ICBM-placenta 37.09 Fibroblast-MSC 39.63ICBM-Umbilical 40.15 Fibroblast-Umbilical 41.59 MSC-Placenta 42.84MSC-Umbilical 46.86 ICBM-placenta 48.41

Tables 8-3, 8-4, and 8-5 show the expression of genes increased inplacenta-derived cells (Table 8-3), increased in umbilical cord-derivedcells (Table 8-4), and reduced in umbilical cord and placenta-derivedcells (Table 8-5).

TABLE 8-3 Genes which are specifically increased in expression in theplacenta-derived cells as compared to the other cell lines assayed. NCBIAccession Probe Set ID Gene Name Number 209732_at C-type (calciumdependent, carbohydrate- AF070642 recognition domain) lectin,superfamily member 2 (activation-induced) 206067_s_at Wilms tumor 1NM_024426 207016_s_at aldehyde dehydrogenase 1 family, member AB015228A2 206367_at Renin NM_000537 210004_at oxidized low density lipoproteinAF035776 (lectin-like) receptor 1 214993_at Homo sapiens, clone IMAGE:4179671, AF070642 mRNA, partial cds 202178_at protein kinase C, zetaNM_002744 209780_at hypothetical protein DKFZp564F013 AL136883 204135_atdownregulated in ovarian cancer 1 NM_014890 213542_at Homo sapiens mRNA;cDNA AI246730 DKFZp547K1113 (from clone DKFZp547K1113)

TABLE 8-4 Genes which are specifically increased in expression inumbilical cord- derived cells as compared to the other cell linesassayed. NCBI Accession Probe Set ID Gene Name Number 202859_x_atInterleukin 8 NM_000584 211506_s_at Interleukin 8 AF043337 210222_s_atreticulon 1 BC000314 204470 _at chemokine (C-X-C motif) ligand 1NM_001511 (melanoma growth stimulating activity 206336_at chemokine(C-X-C motif) ligand 6 NM_002993 (granulocyte chemotactic protein 2)207850_at Chemokine (C-X-C motif) ligand 3 NM_002090 203485_at reticulon1 NM_021136 202644_s_at tumor necrosis factor, alpha-induced NM_006290protein 3

TABLE 8-5 Genes which were decreased in expression in the umbilical cordand placenta cells as compared to the other cell lines assayed. ProbeSet ID Gene name NCBI Accession # 210135_s_at short stature homeobox 2AF022654.1 205824_at heat shock 27 kDa protein 2 NM_001541.1 209687_atchemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1)U19495.1 203666_at chemokine (C-X-C motif) ligand 12 (stromalcell-derived factor 1) NM_000609.1 212670_at elastin (supravalvularaortic stenosis, Williams-Beuren syndrome) AA479278 213381_at Homosapiens mRNA; cDNA DKFZp586M2022 (from clone N91149 DKFZp586M2022)206201_s_at mesenchyme homeobox 2 (growth arrest-specific homeobox)NM_005924.1 205817_at Sine oculis homeobox homolog 1 (Drosophila)NM_005982.1 209283_at crystallin, alpha B AF007162.1 212793_atdishevelled associated activator of morphogenesis 2 BF513244 213488_atDKFZP586B2420 protein AL050143.1 209763_at similar to neuralin 1AL049176 205200_at Tetranectin (plasminogen binding protein) NM_003278.1205743_at src homology three (SH3) and cysteine rich domain NM_003149.1200921_s_at B-cell translocation gene 1, anti-proliferative NM_001731.1206932_at cholesterol 25-hydroxylase NM_003956.1 204198_s_atrunt-related transcription factor 3 AA541630 219747_at hypotheticalprotein FLJ23191 NM_024574.1 204773_at Interleukin 11 receptor, alphaNM_004512.1 202465_at Procollagen C-endopeptidase enhancer NM_002593.2203706_s_at Frizzled homolog 7 (Drosophila) NM_003507.1 212736_athypothetical gene BC008967 BE299456 214587_at Collagen, type VIII, alpha1 BE877796 201645_at Tenascin C (hexabrachion) NM_002160.1 210239_atiroquois homeobox protein 5 U90304.1 203903_s_at Hephaestin NM_014799.1205816_at integrin, beta 8 NM_002214.1 203069_at synaptic vesicleglycoprotein 2 NM_014849.1 213909_at Homo sapiens cDNA FLJ12280 fis,clone MAMMA1001744 AU147799 206315_at cytokine receptor-like factor 1NM_004750.1 204401_at potassium intermediate/small conductancecalcium-activated channel, NM_002250.1 subfamily N, member 4 216331_atintegrin, alpha 7 AK022548.1 209663_s_at integrin, alpha 7 AF072132.1213125_at DKFZP586L151 protein AW007573 202133_at transcriptionalco-activator with PDZ-binding motif (TAZ) AA081084 206511_s_at Sineoculis homeobox homolog 2 (Drosophila) NM_016932.1 213435_at KIAA1034protein AB028957.1 206115_at early growth response 3 NM_004430.1213707_s_at distal-less homeobox 5 NM_005221.3 218181_s_at hypotheticalprotein FLJ20373 NM_017792.1 209160_at aldo-keto reductase family 1,member C3 (3-alpha hydroxysteroid AB018580.1 dehydrogenase, type II)213905_x_at Biglycan AA845258 201261_x_at Biglycan BC002416.1 202132_attranscriptional co-activator with PDZ-binding motif (TAZ) AA081084214701_s_at fibronectin 1 AJ276395.1 213791_at Proenkephalin NM_006211.1205422_s_at Integrin, beta-like 1 (with EGF-like repeat domains)NM_004791.1 214927_at Homo sapiens mRNA full length insert cDNA cloneEUROIMAGE AL359052.1 1968422 206070_s_at EphA3 AF213459.1 212805_atKIAA0367 protein AB002365.1 219789_at natriuretic peptide receptorC/guanylate cyclase C (atrionatriuretic AI628360 peptide receptor C)219054_at hypothetical protein FLJ14054 NM_024563.1 213429_at Homosapiens mRNA; cDNA DKFZp564B222 (from clone AW025579 DKFZp564B222)204929_s_at vesicle-associated membrane protein 5 (myobrevin)NM_006634.1 201843_s_at EGF-containing fibulin-like extracellular matrixprotein 1 NM_004105.2 221478_at BCL2/adenovirus E1B 19 kDa interactingprotein 3-like AL132665.1 201792_at AE binding protein 1 NM_001129.2204570_at cytochrome c oxidase subunit VIIa polypeptide 1 (muscle)NM_001864.1 201621_at neuroblastoma, suppression of tumorigenicity 1NM_005380.1 202718_at Insulin-like growth factor binding protein 2, 36kDa NM_000597.1

Tables 8-6, 8-7, and 8-8 show the expression of genes increased in humanfibroblasts (Table 8-6), ICBM cells (Table 8-7), and MSCs (Table 8-8).

TABLE 8-6 Genes which were increased in expression in fibroblasts ascompared to the other cell lines assayed. dual specificity phosphatase 2KIAA0527 protein Homo sapiens cDNA: FLJ23224 fis, clone ADSU02206dynein, cytoplasmic, intermediate polypeptide 1 ankyrin 3, node ofRanvier (ankyrin G) inhibin, beta A (activin A, activin AB alphapolypeptide) ectonucleotide pyrophosphatase/phosphodiesterase 4(putative function) KIAA1053 protein microtubule-associated protein 1Azinc finger protein 41 HSPC019 protein Homo sapiens cDNA: FLJ23564 fis,clone LNG10773 Homo sapiens mRNA; cDNA DKFZp564A072 (from cloneDKFZp564A072) LIM protein (similar to rat protein kinase C-bindingenigma) inhibitor of kappa light polypeptide gene enhancer in B-cells,kinase complex-associated protein hypothetical protein FLJ22004 Human(clone CTG-A4) mRNA sequence ESTs, Moderately similar to cytokinereceptor-like factor 2; cytokine receptor CRL2 precursor [Homo sapiens]transforming growth factor, beta 2 hypothetical protein MGC29643 antigenidentified by monoclonal antibody MRC OX-2 putative X-linked retinopathyprotein

TABLE 8-7 Genes which were increased in expression in the ICBM-derivedcells as compared to the other cell lines assayed. cardiac ankyrinrepeat protein MHC class I region ORF integrin, alpha 10 hypotheticalprotein FLJ22362 UDP-N-acetyl-alpha-D-galactosamine:polypeptideN-acetylgalactosaminyltransferase 3 (GalNAc-T3) interferon-inducedprotein 44 SRY (sex determining region Y)-box 9 (campomelic dysplasia,autosomal sex-reversal) keratin associated protein 1-1 hippocalcin-like1 jagged 1 (Alagille syndrome) proteoglycan 1, secretory granule

TABLE 8-8 Genes which were increased in expression in the MSC cells ascompared to the other cell lines assayed. interleukin 26maltase-glucoamylase (α-glucosidase) nuclear receptor subfamily 4, groupA, member 2 v-fos FBJ murine osteosarcoma viral oncogene homologhypothetical protein DC42 nuclear receptor subfamily 4, group A, member2 FBJ murine osteosarcoma viral oncogene homolog B WNT1 induciblesignaling pathway protein 1 MCF.2 cell line derived transformingsequence potassium channel, subfamily K, member 15 cartilagepaired-class homeoprotein 1 Homo sapiens cDNA FLJ12232 fis, cloneMAMMA1001206 Homo sapiens cDNA FLJ34668 fis, clone LIVER2000775 jun Bproto-oncogene B-cell CLL/lymphoma 6 (zinc finger protein 51) zincfinger protein 36, C3H type, homolog (mouse)

This example was performed to provide a molecular characterization ofthe cells derived from umbilical cord and placenta. This analysisincluded cells derived from three different umbilical cords and threedifferent placentas. The study also included two different lines ofdermal fibroblasts, three lines of mesenchymal stem cells, and threelines of iliac crest bone marrow cells. The mRNA that was expressed bythese cells was analyzed on a GENECHIP oligonucleotide array thatcontained oligonucleotide probes for 22,000 genes.

The analysis revealed that transcripts for 290 genes were present indifferent amounts in these five different cell types. These genesinclude ten genes that are specifically increased in theplacenta-derived cells and seven genes specifically increased in theumbilical cord-derived cells. Fifty-four genes were found to havespecifically lower expression levels in placenta-derived and umbilicalcord tissue-derived cells.

Example 9 Immunohistochemical Characterization of Cellular Phenotypes

The phenotypes of cells found within human umbilical cord tissue wereanalyzed by immunohistochemistry.

Human umbilical cord tissue was harvested and immersion fixed in 4%(w/v) paraformaldehyde overnight at 4° C. Immunohistochemistry wasperformed using antibodies directed against the following epitopes (seeTable 8-1): vimentin (1:500; Sigma, St. Louis, Mo.), desmin (1:150,raised against rabbit; Sigma; or 1:300, raised against mouse; Chemicon,Temecula, Calif.), alpha-smooth muscle actin (SMA; 1:400; Sigma),cytokeratin 18 (CK18; 1:400; Sigma), von Willebrand Factor (vWF; 1:200;Sigma), and CD34 (human CD34 Class III; 1:100; DAKOCytomation,Carpinteria, Calif.). In addition, the following markers were tested:anti-human GROalpha-PE (1:100; Becton Dickinson, Franklin Lakes, N.J.),anti-human GCP-2 (1:100; Santa Cruz Biotech, Santa Cruz, Calif.),anti-human oxidized LDL receptor 1 (ox-LDL R1; 1:100; Santa CruzBiotech), and anti-human NOGO-A (1:100; Santa Cruz Biotech). Fixedspecimens were trimmed with a scalpel and placed within OCT embeddingcompound (Tissue-Tek OCT; Sakura, Torrance, Calif.) on a dry ice bathcontaining ethanol. Frozen blocks were then sectioned (10 μm thick)using a standard cryostat (Leica Microsystems) and mounted onto glassslides for staining

Immunohistochemistry was performed similar to previous studies. (E.g.,Messina et al., Exper. Neurol., 2003; 184: 816-829). Tissue sectionswere washed with phosphate-buffered saline (PBS) and exposed to aprotein blocking solution containing PBS, 4% (v/v) goat serum (Chemicon,Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100; Sigma) for 1hour to access intracellular antigens. In instances where the epitope ofinterest would be located on the cell surface (CD34, ox-LDL R1), tritonwas omitted in all steps of the procedure in order to prevent epitopeloss. Furthermore, in instances where the primary antibody was raisedagainst goat (GCP-2, ox-LDL R1, NOGO-A), 3% (v/v) donkey serum was usedin place of goat serum throughout the procedure. Primary antibodies,diluted in blocking solution, were then applied to the sections for aperiod of 4 hours at room temperature. Primary antibody solutions wereremoved, and cultures washed with PBS prior to application of secondaryantibody solutions (1 hour at room temperature) containing block alongwith goat anti-mouse IgG-Texas Red (1:250; Molecular Probes, Eugene,Oreg.) and/or goat anti-rabbit IgG-Alexa 488 (1:250; Molecular Probes)or donkey anti-goat IgG-FITC (1:150; Santa Cruz Biotech). Cultures werewashed, and 10 micromolar DAPI (Molecular Probes) was applied for 10minutes to visualize cell nuclei.

Following immune-staining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epifluorescentmicroscope (Olympus, Melville, N.Y.). Positive staining was representedby fluorescence signal above control staining. Representative imageswere captured using a digital color video camera and ImagePro software(Media Cybernetics, Carlsbad, Calif.). For triple-stained samples, eachimage was taken using only one emission filter at a time. Layeredmontages were then prepared using Adobe Photoshop software (Adobe, SanJose, Calif.).

TABLE 9-1 Summary of Primary Antibodies Used Antibody ConcentrationVendor Vimentin 1:500 Sigma, St. Louis, MO Desmin (rb) 1:150 SigmaDesmin (m) 1:300 Chemicon, Temecula, CA alpha-smooth muscle 1:400 Sigmaactin (SMA) Cytokeratin 18 (CK18) 1:400 Sigma von Willebrand factor1:200 Sigma (vWF) CD34 III 1:100 DakoCytomation, Carpinteria, CAGROalpha-PE 1:100 BD, Franklin Lakes, NJ GCP-2 1:100 Santa Cruz BiotechOx-LDL R1 1:100 Santa Cruz Biotech NOGO-A 1:100 Santa Cruz Biotech

Vimentin, desmin, SMA, CK18, vWF, and CD34 markers were expressed in asubset of the cells found within umbilical cord (data not shown). Inparticular, vWF and CD34 expression were restricted to blood vesselscontained within the cord. CD34 positive (CD34⁺) cells were on theinnermost layer (lumen side). Vimentin expression was found throughoutthe matrix and blood vessels of the cord. SMA was limited to the matrixand outer walls of the artery and vein, but not contained within thevessels themselves. CK18 and desmin were observed within the vesselsonly, desmin being restricted to the middle and outer layers.

None of these markers were observed within umbilical cord (data notshown).

Vimentin, desmin, alpha-smooth muscle actin, cytokeratin 18, vonWillebrand Factor, and CD 34 are expressed in cells within humanumbilical cord. Based on in vitro characterization studies showing thatonly vimentin and alpha-smooth muscle actin are expressed, the datasuggests that the current process of umbilical cord-derived cellisolation harvests a subpopulation of cells or that the cells isolatedchange expression of markers to express vimentin and alpha-smooth muscleactin.

Example 10 Secretion of Trophic Factors

The secretion of selected trophic factors from UTC was measured. Factorswere selected that have angiogenic activity e.g., hepatocyte growthfactor (HGF) (Rosen et al., Ciba Found. Symp., 1997; 212:215-26);monocyte chemotactic protein 1 (MCP-1) (Salcedo et al., Blood, 2000; 96;34-40); interleukin-8 (IL-8) (Li et al., J. Immunol., 2003;170:3369-76); keratinocyte growth factor (KGF); basic fibroblast growthfactor (bFGF); vascular endothelial growth factor (VEGF) (Hughes et al.,Ann. Thorac. Surg. 2004; 77:812-8); tissue inhibitor of matrixmetalloproteinase 1 (TIMP1); angiopoietin 2 (ANG2); platelet derivedgrowth factor (PDGFbb); thrombopoietin (TPO); heparin-binding epidermalgrowth factor (HB-EGF); stromal-derived factor 1 alpha (SDF-1alpha),neurotrophic/neuroprotective activity (brain-derived neurotrophic factor(BDNF) (Cheng et al., Dev. Biol., 2003; 258; 319-33); interleukin-6(IL-6); granulocyte chemotactic protein-2 (GCP-2); transforming growthfactor beta2 (TGFbeta2)); or chemokine activity (macrophage inflammatoryprotein 1 alpha (MIP1alpha); macrophage inflammatory protein 1 beta(MIP1beta); monocyte chemoattractant-1 (MCP-1); Rantes (regulated onactivation, normal T cell expressed and secreted); 1309; thymus andactivation-regulated chemokine (TARC); Eotaxin; macrophage-derivedchemokine (MDC); and (IL-8).

Cells derived from umbilical cord, as well as human fibroblasts derivedfrom human neonatal foreskin, were cultured in growth medium ongelatin-coated T75 flasks. Cells were cryopreserved at passage 11 andstored in liquid nitrogen. After thawing, growth medium was added to thecells, followed by transfer to a 15 ml centrifuge tube andcentrifugation of the cells at 150×g for 5 minutes. The cell pellet wasresuspended in 4 ml growth medium, and the cells were counted. Cellswere seeded at 5,000 cells/cm² in T75 flasks each containing 15 ml ofgrowth medium, and cultured for 24 hours. The medium was changed to aserum-free medium (DMEM-low glucose (Gibco), 0.1% (w/v) bovine serumalbumin (Sigma), penicillin (50 U/ml) and streptomycin (50 μg/ml,Gibco)) for 8 hours. Conditioned serum-free medium was collected at theend of incubation by centrifugation at 14,000×g for 5 minutes and storedat −20° C.

To estimate the number of cells in each flask, the cells were washedwith phosphate-buffered saline (PBS) and detached using 2 mltrypsin/EDTA (Gibco). Trypsin activity was inhibited by addition of 8 mlgrowth medium. The cells were centrifuged at 150×g for 5 minutes. Thesupernatant was removed, and the cells were resuspended in 1 ml GrowthMedium. The cell number was estimated with a hemocytometer.

Cells were grown at 37° C. in 5% CO₂ and atmospheric oxygen. The amountof MCP-1, IL-6, VEGF, SDF-1alpha, GCP-2, IL-8, and TGF-beta2 produced byeach cell sample was determined by ELISA (R&D Systems, Minneapolis,Mn.). All assays were performed according to the manufacturer'sinstructions. Values presented are picograms per ml per million cells(n=2, sem).

Chemokines (MIP1alpha, MIP1beta, MCP-1, Rantes, 1309, TARC, Eotaxin,MDC, IL8), BDNF, and angiogenic factors (HGF, KGF, bFGF, VEGF, TIMP1,ANG2, PDGFbb, TPO, HB-EGF were measured using SearchLight ProteomeArrays (Pierce Biotechnology Inc.). The Proteome Arrays are multiplexedsandwich ELISAs for the quantitative measurement of two to sixteenproteins per well. The arrays are produced by spotting a 2×2, 3×3, or4×4 pattern of four to sixteen different capture antibodies into eachwell of a 96-well plate. Following a sandwich ELISA procedure, theentire plate is imaged to capture the chemiluminescent signal generatedat each spot within each well of the plate. The signal generated at eachspot is proportional to the amount of target protein in the originalstandard or sample.

MCP-1 and IL-6 were secreted by umbilicus-derived PPDCs and dermalfibroblasts (Table 10-1). SDF-1alpha and GCP-2 were secreted byfibroblasts. GCP-2 and IL-8 were secreted by umbilicus-derived PPDCs.TGF-beta2 was not detected from either cell type by ELISA.

TABLE 10-1 ELISA Results: Detection of Trophic Factors MCP-1 IL-6 VEGFSDF-1α GCP-2 IL-8 TGF-beta2 Fibroblast  17 ± 1 61 ± 3 29 ± 2 19 ± 1 21 ±1 ND ND Umbilical (022803) 1150 ± 74 4234 ± 289 ND ND 160 ± 11 2058 ±145 ND Umbilical (071003) 2794 ± 84 1356 ± 43  ND ND 2184 ± 98  2369 ±23  ND Key: ND: Not Detected., =/− sem

Searchlight™ Multiplexed ELISA assay. TIMP1, TPO, KGF, HGF, FGF, HBEGF,BDNF, MIP1beta, MCPJ, RANTES, 1309, TARC, MDC, and IL-8 were secretedfrom umbilicus-derived PPDCs (Tables 10-2 and 10-3). No Ang2, VEGF, orPDGFbb were detected.

TABLE 10-2 Searchlight ™ Multiplexed ELISA assay results TIMP1 ANG2PDGFbb TPO KGF HGF FGF VEGF HBEGF BDNF hFB 19306.3 ND ND 230.5 5.0 ND ND27.9 1.3 ND U1 57718.4 ND ND 1240.0 5.8 559.3 148.7 ND 9.3 165.7 U321850.0 ND ND 1134.5 9.0 195.6 30.8 ND 5.4 388.6 Key: hFB (humanfibroblasts), U1 (umbilicus-derived PPDC (022803)), U3(umbilicus-derived PPDC (071003)), ND: Not Detected.

TABLE 10-3 Searchlight ™ Multiplexed ELISA assay results MIP1a MIP1bMCP1 RANTES I309 TARC Eotaxin MDC IL8 hFB ND ND 39.6 ND ND 0.1 ND ND204.9 U1 ND 8.0 1694.2 ND 22.4 37.6 ND 18.9 51930.1 U3 ND 5.2 2018.741.5 11.6 21.4 ND 4.8 10515.9 Key: hFB (human fibroblasts), U1(umbilicus-derived PPDC (022803)), U3 (umbilicus-derived PPDC (071003)),ND: Not Detected

Umbilicus-derived cells secreted a number of trophic factors. Some ofthese trophic factors, such as HGF, bFGF, MCP-1 and IL-8, play importantroles in angiogenesis. Other trophic factors, such as BDNF and IL-6,have important roles in neural regeneration or protection.

Example 11 Assay for Telomerase Activity

Telomerase functions to synthesize telomere repeats that serve toprotect the integrity of chromosomes and to prolong the replicative lifespan of cells (Liu, K, et al., PNAS, 1999; 96:5147-5152). Telomeraseconsists of two components, telomerase RNA template (hTER) andtelomerase reverse transcriptase (hTERT). Regulation of telomerase isdetermined by transcription of hTERT but not hTER. Real-time polymerasechain reaction (PCR) for hTERT mRNA thus is an accepted method fordetermining telomerase activity of cells.

Cell Isolation

Real-time PCR experiments were performed to determine telomeraseproduction of human umbilical cord tissue-derived cells. Human umbilicalcord tissue-derived cells were prepared in accordance with the aboveExamples and the examples set forth in U.S. Pat. No. 7,510,873.Generally, umbilical cords obtained from National Disease ResearchInterchange (Philadelphia, Pa.) following a normal delivery were washedto remove blood and debris and mechanically dissociated. The tissue wasthen incubated with digestion enzymes including collagenase, dispase,and hyaluronidase in culture medium at 37° C. Human umbilical cordtissue-derived cells were cultured according to the methods set forth inthe examples of the '012 application. Mesenchymal stem cells and normaldermal skin fibroblasts (cc-2509 lot #9F0844) were obtained fromCambrex, Walkersville, Md. A pluripotent human testicular embryonalcarcinoma (teratoma) cell line nTera-2 cells (NTERA-2 cl.D1) (See, Plaiaet al., Stem Cells, 2006; 24(3):531-546) was purchased from ATCC(Manassas, Va.) and was cultured according to the methods set forth inU.S. Pat. No. 7,510,873.

Total RNA Isolation

RNA was extracted from the cells using RNeasy® kit (Qiagen, Valencia,Calif.). RNA was eluted with 50 μl DEPC-treated water and stored at −80°C. RNA was reverse transcribed using random hexamers with the TaqMan®reverse transcription reagents (Applied Biosystems, Foster City, Calif.)at 25° C. for 10 minutes, 37° C. for 60 minutes and 95° C. for 10minutes. Samples were stored at −20° C.

Real-time PCR

PCR was performed on cDNA samples using the Applied BiosystemsAssays-On-Demand™ (also known as TaqMan® Gene Expression Assays)according to the manufacturer's specifications (Applied Biosystems).This commercial kit is widely used to assay for telomerase in humancells. Briefly, hTert (human telomerase gene) (Hs00162669) and humanGAPDH (an internal control) were mixed with cDNA and TaqMan® UniversalPCR master mix using a 7000 sequence detection system with ABI prism7000 SDS software (Applied Biosystems). Thermal cycle conditions wereinitially 50° C. for 2 minutes and 95° C. for 10 minutes followed by 40cycles of 95° C. for 15 seconds and 60° C. for 1 minute. PCR data wasanalyzed according to the manufacturer's specifications.

Human umbilical cord tissue-derived cells (ATCC Accession No. PTA-6067),fibroblasts, and mesenchymal stem cells were assayed for hTert and 18SRNA. As shown in Table 11-1, hTert, and hence telomerase, was notdetected in human umbilical cord tissue-derived cells.

TABLE 11-1 hTert 18S RNA Umbilical cells (022803) ND + Fibroblasts ND +ND - not detected; + signal detected

Human umbilical cord tissue-derived cells (isolate 022803, ATCCAccession No. PTA-6067) and nTera-2 cells were assayed and the resultsshowed no expression of the telomerase in two lots of human umbilicalcord tissue-derived cells while the teratoma cell line revealed highlevel of expression (Table 11-2).

TABLE 11-2 hTert GAPDH Cell type Exp. 1 Exp. 2 Exp. 1 Exp. 2 hTert normnTera2 25.85 27.31 16.41 16.31 0.61 022803 — — 22.97 22.79 —

Therefore, it can be concluded that the human umbilical tissue-derivedcells as disclosed herein do not express telomerase.

While the invention has been described and illustrated herein byreferences to various specific materials, procedures and examples, it isunderstood that the invention is not restricted to the particularcombinations of material and procedures selected for that purpose.Numerous variations of such details can be implied as will beappreciated by those skilled in the art. It is intended that thespecification and examples be considered as exemplary, only, with thetrue scope and spirit of the invention being indicated by the followingclaims. All references, patents, and patent applications referred to inthis application are herein incorporated by reference in their entirety.

What is claimed is:
 1. A method of determining the pharmacokineticprofile of human umbilical cord tissue-derived cells in patient: a.administering a known quantity of a homogeneous population of isolatedhuman umbilical cord tissue-derived cells of either XX or XY karyotype;b. providing a blood sample from a male or female patient that has beentreated with the known quantity of human umbilical cord tissue-derivedcells; c. testing the patient blood sample to detect one or more uniquemarkers positive for the human umbilical cord tissue-derived cells andCD45; d. comparing the test results to a unique marker profile for thehuman umbilical cord tissue-derived cells and CD45 to distinguishbetween the patient peripheral blood mononuclear cells and humanumbilical cord tissue-derived cells based on the detection of the uniquemarker profile; e. quantifying the number of human umbilical cordtissue-derived cells; and f. generating a pharmacokinetic profile forthe treatment using the homogeneous population of isolated humanumbilical cord tissue-derived cells, wherein the unique marker profileof the human umbilical cord tissue-derived cells comprises one or moremarkers positive for the human umbilical cord tissue-derived cells,wherein the unique marker profile of the human umbilical cordtissue-derived cells comprises one or more markers positive for thehuman umbilical cord tissue-derived cells, wherein said one or moremarkers positive for the human umbilical cord tissue derived cells donot comprise CD45, and wherein the human umbilical cord tissue-derivedcells are isolated from human umbilical cord tissue substantially freeof blood, are capable of self-renewal and expansion in culture, have thepotential to differentiate, and have the following characteristics: (1)express CD10, CD13, CD44, CD90, NRP1, DKK3, LAMP1, and HLA-ABC; (2) donot express CD31, CD34, HLA-DR and CD117, and (3) do not express hTERTor telomerase.
 2. The method of claim 1, further comprising performingan enrichment step between steps (b) and (c).
 3. The method of claim 2wherein the enrichment step is magnetic capture technology.
 4. Themethod of claim 1, wherein the patient is a human, non-human primate,mouse, rat, hamster, guinea pig, dog, or pig.